WO1997035014A1 - A KINASE CAPABLE OF SITE-SPECIFIC PHOSPHORYLATION OF IλB$g(a) - Google Patents
A KINASE CAPABLE OF SITE-SPECIFIC PHOSPHORYLATION OF IλB$g(a) Download PDFInfo
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- WO1997035014A1 WO1997035014A1 PCT/US1997/004195 US9704195W WO9735014A1 WO 1997035014 A1 WO1997035014 A1 WO 1997035014A1 US 9704195 W US9704195 W US 9704195W WO 9735014 A1 WO9735014 A1 WO 9735014A1
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- kinase
- iκbα
- nucleic acid
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/81—Packaged device or kit
Definitions
- the present invention relates, in general, to a kinase which in its activated state is capable of site-specific phosphorylation of I ⁇ B ⁇ , I ⁇ B ⁇ kinase.
- the present invention relates to the purified kinase, purified polypeptide subunits ofthe kinase, nucleic acid molecules coding for the purified polypeptide subunits; recombinant nucleic acid molecules; cells containing the recombinant nucleic acid molecules; antibodies having binding affinity specifically to the kinase or its polypeptide subunits; hybridomas containing the antibodies; nucleic acid probes for the detection ofthe nucleic acid encoding the kinase; a method of detecting nucleic acids encoding the kinase or polypeptides ofthe kinase in a sample; and kits containing nucleic acid probes or antibodies.
- This invention further relates to bioassays using the nucleic acid sequence, protein or antibodies of this invention to diagnose, assess, or prognose a mammal afflicted with an undesired activation of NF- ⁇ B.
- This invention also relates to ligands, agonists, and antagonists of the kinase, and diagnostic and therapeutic uses thereof.
- This invention also relates to bioassays using the kinase or polypeptides of the kinase of this invention to identify ligands, agonists, and antagonists. More specifically, this invention relates to selective inhibitors ofthe kinase and to structure-based design of ligands, agonists, and antagonists ofthe kinase.
- This invention further relates to gene therapy using the nucleic acids of the invention.
- cytokines e.g., TNF- ⁇ and IL-l ⁇
- viral proteins e.g., HTLV-1 Tax
- antigens e.g., okadaic acid and calyculin A
- UV light e.g., UV light.
- the rel/NF- ⁇ B family of transcriptional activator proteins plays an essential role in the signal transduction pathways that link these signals to gene activation (reviewed by Siebenlist, U. et al., Annu. Rev. Cell. Biol.
- I ⁇ B ⁇ or I ⁇ B ⁇ members of the I ⁇ B family of inhibitor proteins.
- I ⁇ B ⁇ and most likely I ⁇ B ⁇
- stimulation of cells leads to rapid phosphorylation and degradation of the inhibitor. Consequently NF- ⁇ B is released and translocates into the nucleus where it activates the expression of target genes.
- Phosphorylation of I ⁇ B ⁇ per se is not sufficient to dissociate NF- ⁇ B from the latent complex (Palombella, V.J., Cell. 75:773-785 (1994); Traenckner, E.B.-M. et ai. EMBOJ. 73:5433-56441 (1994); Finco, T.S. et al. Proc. Natl. Acad. Sci.
- Ubiquitination is a three-step process: First, ubiquitin is activated by a ubiquitin activating enzyme (El); the activated ubiquitin is then transferred to a ubiquitin carrier protein (E2, also referred to as ubiquitin conjugating enzyme or UBC); finally, ubiquitin is conjugated to a protein substrate by forming an isopeptide bond between the carboxyl terminal glycine residue of ubiquitin and the ⁇ -amino group of one or more lysine residues ofthe protein substrate. This conjugation step often requires a ubiquitin protein ligase (E3).
- E3 ubiquitin protein ligase
- ubiquitin can be ligated to a protein substrate to form multi-ubiquitin chains, which are then recognized by a large, ATP-dependent protease (MW -2000 kDa) called the 26S proteasome.
- the 26S proteasome is composed of a 20S catalytic core, and a 19S regulatory complex (reviewed by Goldberg, A.L., Science 268:522-523 (1995)).
- Multiple E2s and E3s function together to mediate the ubiquitination of a variety of cellular proteins. For example, there are at least a dozen E2s in yeast that display distinct substrate specificities and carry out distinct cellular functions.
- UBC4/UBC5 mediate the ubiquitination ofthe P53 protein in conjunction with the HPV-16 E6-E6AP complex, which functions as an E3 (Schaffner, M. et al, Cell. 75:495-505 (1993)).
- E2s have also been implicated in the ubiquitination ofthe MAT ⁇ 2 Processor (Chen, P. et al, Cell 74:357-369 (1993)), cyclin B (King, R.W. et al, Cell 57:279-288 (1995)), and the NF- ⁇ B precursor protein P105 (Orian, A. et al, J. Biol. Chem. 270:21707-21714 (1995)).
- PLC protein kinase C
- HRI heme- regulated eIF-2 ⁇ kinase
- Ghosh & Baltimore protein kinase A
- ROIs reactive oxygen intermediates
- the present invention involves fractionating HeLa cell cytoplasmic extracts and assaying for specific phosphorylation and ubiquitination of I ⁇ B ⁇ .
- the present invention provides a high molecular weight kinase complex that, in its activated state, specifically phosphorylates I ⁇ B ⁇ at serines 32 and 36.
- ubiquitin, and El are not only involved in the ubiquitination of I ⁇ B ⁇ , but may also be required for the phosphorylation of I ⁇ B ⁇ .
- I ⁇ B ⁇ kinase can be activated by a prior ubiquitination event.
- ubiquitination serves a regulatory function without involving proteolysis.
- this I ⁇ B ⁇ may be activated by MEKK1.
- the present invention provides a substantially purified large, multi-subunit kinase (MW -700 kDa) that, in its active state, phosphorylates I ⁇ B ⁇ at serines 32 and 36.
- the kinase comprises an amino acid sequence which is at least 60% homologous to the amino acid sequence of any one of Figures 21 A-D.
- this kinase may be activated by a ubiquitination event requiring the ubiquitin activating enzyme (El), a specific ubiquitin carrier protein (E2) ofthe UBC4/UBC5 family, and ubiquitin.
- El ubiquitin activating enzyme
- E2 a specific ubiquitin carrier protein
- ubiquitination serves a novel regulatory function that does not involve proteolysis.
- the kinase may be activated via phosphorylation by MEKK-1. Additional activation routes, e.g. phosphorylation by a kinase other than MEKK-1 , may also be possible.
- the invention further provides the substantially pure polypeptide subunits ofthe above-described kinase.
- the invention provides isolated nucleic acid molecules coding for subunits ofthe above-described kinase.
- the invention also provides a nucleic acid probe for the specific detection of the presence of nucleic acid encoding the above-described kinase or its subunits or a fragment thereof in a sample.
- the invention further provides a method of detecting the above-described nucleic acid in a sample.
- the invention also provides a kit for detecting the presence ofthe above- described nucleic acid in a sample.
- the invention further provides a recombinant nucleic acid molecule comprising, 5' to 3', a promoter effective to initiate transcription in a host cell and the above-described nucleic acid molecule.
- the invention also provides a recombinant nucleic acid molecule comprising a vector and the above-described nucleic acid molecule.
- the invention also provides a cell that contains the above-described recombinant nucleic acid molecule.
- the invention further provides a non-human organism that contains the above-described recombinant nucleic acid molecule.
- the invention also provides an antibody having binding affinity specifically to the above-described kinase or to a subunit of the above-described kinase.
- the invention further provides a method of detecting the above-described kinase or one of its subunits in a sample.
- the invention also provides a method of measuring the amount of the above-described kinase in a sample.
- the invention further provides a diagnostic kit comprising a first container means containing the above-described antibody, and a second container means containing a conjugate comprising a binding partner ofthe monoclonal antibody and a label.
- the invention also provides a hybridoma which produces the above- described monoclonal antibody.
- the invention further provides diagnostic methods for human disease, in particular diseases, disorders, and injuries resulting from an undesired activation of NF-KB.
- the invention also provides methods for therapeutic uses involving (1 ) the nucleic acid sequence encoding the above-described kinase or a subunit thereof and/or (2) the above-described kinase or a subunit thereof.
- the invention provides ligands, agonists, and antagonists of the above- described kinase and diagnostic and therapeutic uses for these molecules.
- the molecule is a selective inhibitor of kinase activity, i.e. the ability to phosphorylate I ⁇ B ⁇ at serine residues 32 and 36.
- the invention also provides assays for the identification of ligands, agonists and antagonists ofthe above-described kinase.
- rDNA DNA
- protein purification protein purification
- diagnostic and therapeutic methods are extensively utilized.
- rDNA DNA
- isolated nucleic acid molecule refers to a polymer of nucleotides, and includes but should not be limited to DNA and RNA.
- a DNA segment refers to a molecule comprising a linear stretch of nucleotides wherein the nucleotides are present in a sequence that can encode, through the genetic code, a molecule comprising a linear sequence of amino acid residues that is referred to as a protein, a protein fragment or a polypeptide.
- a DNA sequence related to a single polypeptide chain or protein, and as used herein includes the 5' and 3' untranslated ends.
- the polypeptide can be encoded by a full-length sequence or any portion ofthe coding sequence, so long as the functional activity ofthe protein is retained.
- cDNA Complementary DNA
- mRNA messenger RNA
- Structural Gene A DNA sequence that is transcribed into mRNA that is then translated into a sequence of amino acids characteristic of a specific polypeptide.
- Restriction Endonuclease A restriction endonuclease (also restriction enzyme) is an enzyme that has the capacity to recognize a specific base sequence
- DNA molecule at every place where this sequence appears.
- EcoRI recognizes the base sequence GAATTC/CTTAAG.
- restriction fragments The DNA molecules produced by digestion with a restriction endonuclease are referred to as restriction fragments. Any given genome can be digested by a particular restriction endonuclease into a discrete set of restriction fragments.
- Polyacrylamide Gel Electrophoresis PA GE
- PA GE Polyacrylamide Gel Electrophoresis
- the most commonly used technique (though not the only one) for achieving a fractionation of polypeptides on the basis of size is polyacrylamide gel electrophoresis.
- the principle of this method is that polypeptide molecules migrate through the gel as though it were a sieve that retards the movement ofthe largest molecules to the greatest extent and the movement of the smallest molecules to the least extent.
- polypeptide fragment the greater the mobility under electrophoresis in the polyacrylamide gel.
- polypeptides typically are continuously exposed to the detergent sodium dodecyl sulfate (SDS). under which conditions the polypeptides are denatured. Native gels are run in the absence of SDS.
- SDS detergent sodium dodecyl sulfate
- polypeptides fractionated by polyacrylamide gel electrophoresis can be visualized directly by a staining procedure if the number of polypeptide components is small.
- the purpose of the Western transfer procedure (also referred to as blotting) is to physically transfer polypeptides fractionated by polyacrylamide gel electrophoresis onto a nitrocellulose filter paper or another appropriate surface or method, while retaining the relative positions of polypeptides resulting from the fractionation procedure. The blot is then probed with an antibody that specifically binds to the polypeptide of interest.
- Nucleic Acid Hybridization Nucleic acid hybridization depends on the principle that two single-stranded nucleic acid molecules that have complementary base sequences will reform the thermodynamicaliy favored double-stranded structure if they are mixed under the proper conditions.
- the double-stranded structure will be formed between two complementary single- stranded nucleic acids even if one is immobilized on a nitrocellulose filter.
- Southern hybridization procedure the latter situation occurs.
- the DNA ofthe individual to be tested is digested with a restriction endonuclease, fractionated by agarose gel electrophoresis, converted to the single- stranded form, and transferred to nitrocellulose paper, making it available for reannealing to the hybridization probe.
- Antibody Probe To visualize a particular polypeptide sequence in the western blot procedure, a labeled antibody probe is exposed to the fractionated polypeptides bound to the nitrocellulose filter. The areas on the filter that carry polypeptides that bind to the labeled antibody probe become labeled themselves as a consequence ofthe binding. The areas ofthe filter that exhibit such labeling are visualized.
- hybridization conditions can be found in Ausubel, F.M. et al, Current protocols in Molecular Biology, John Wily & Sons, Inc., New York, NY (1989).
- a nitrocellulose filter is incubated overnight at 68°C with labeled probe in a solution containing 50% formamide, high salt (either 5x SSC[20X: 3M NaCl/0.3M trisodium citrate] or 5X SSPE [20X: 3.6M NaCl/0.2M NaH 2 PO ⁇ O.02M EDTA, pH 7.7]), 5X Denhardfs solution, 1% SDS, and 100 ⁇ g/ml denatured salmon sperm DNA.
- Oligonucleotide or Oligomer A molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three. Its exact size will depend on many factors, which in turn depend on the ultimate function or use ofthe oligonucleotide.
- An oligonucleotide can be derived synthetically or by cloning.
- Sequence Amplification A method for generating large amounts of a target sequence.
- one or more amplification primers are annealed to a nucleic acid sequence.
- sequences found adjacent to, or in between the primers are amplified.
- Amplification Primer An oligonucleotide which is capable of annealing adjacent to a target sequence and serving as an initiation point for DNA synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is initiated.
- Vector A plasmid or phage DNA or other DNA sequence into which DNA can be inserted to be cloned.
- the vector can replicate autonomously in a host cell, and can be further characterized by one or a small number of endonuclease recognition sites at which such DNA sequences can be cut in a determinable fashion and into which DNA can be inserted.
- the vector can further contain a marker suitable for use in the identification of cells transformed with the vector. Markers, for example, are tetracydine resistance or ampicillin resistance. The words "cloning vehicle” are sometimes used for "vector.”
- Expression Expression is the process by which a structural gene produces a polypeptide. It involves transcription ofthe gene into mRNA, and the translation of such mRNA into polypeptide(s).
- Expression Vector A vector or vehicle similar to a cloning vector but which is capable of expressing a gene which has been cloned into it, after transformation into a host.
- the cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences.
- Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host and can additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
- a “functional derivative” of a sequence is a molecule that possesses a biological activity (either functional or structural) that is substantially similar to a biological activity ofthe protein or nucleic acid sequence.
- a functional derivative of a protein can contain post-translational modifications such as covalently linked carbohydrate, depending on the necessity of such modifications for the performance of a specific function.
- the term “functional derivative” is intended to include the “fragments,” “segments,” “variants,” “analogs,” or “chemical derivatives” of a molecule.
- a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part ofthe molecule. Such moieties can improve the molecule's solubility, absorption, biological half life, and the like. The moieties can alternatively decrease the toxicity ofthe molecule, eliminate or attenuate any undesirable side effect ofthe molecule, and the like. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980). Procedures for coupling such moieties to a molecule are well known in the art. Variant. A "variant" of a protein or nucleic acid is meant to refer to a molecule substantially similar in structure and biological activity to either the protein or nucleic acid.
- two molecules possess a common activity and can substitute for each other they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the amino acid or nucleotide sequence is not identical.
- Allele is an alternative form of a gene occupying a given locus on the chromosome.
- Mutation is any detectable change in the genetic material which can be transmitted to daughter cells and possibly even to succeeding generations giving rise to mutant cells or mutant individuals. If the descendants of a mutant cell give rise only to somatic cells in multicellular organisms, a mutant spot or area of cells arises. Mutations in the germ line of sexually reproducing organisms can be transmitted by the gametes to the next generation resulting in an individual with the new mutant condition in both its somatic and germ cells.
- a mutation can be any (or a combination of) detectable, unnatural change affecting the chemical or physical constitution, mutability, replication, phenotypic function, or recombination of one or more deoxyribonucleotides; nucleotides can be added, deleted, substituted for, inverted, or transposed to new positions with and without inversion. Mutations can occur spontaneously and can be induced experimentally by application of mutagens or by site-directed mutagenesis. A mutant variation of a nucleic acid molecule results from a mutation. A mutant polypeptide can result from a mutant nucleic acid molecule.
- Species is a group of actually or potentially interbreeding natural populations.
- a species variation within a nucleic acid molecule or protein is a change in the nucleic acid or amino acid sequence that occurs among species and can be determined by DNA sequencing ofthe molecule in question.
- a "purified" protein or nucleic acid is a protein or nucleic acid preparation that is generally free of contaminants, whether produced recombinantly, chemically synthesized or purified from a natural source.
- % homologous When referring to one amino acid sequence as being X% homologous to another amino acid sequence, what is meant is the percentage of sequence identity or sequence similarity. Amino acid sequence similarity is further described in Table 1 , infra.
- Subunit of the Kinase The kinase is a multi-subunit protein. Each subunit is defined herein to be a single polypeptide which is encoded by a nucleic acid sequence. p85, p70, p62, p55, p50, p43, p40, p38, p36, p33, p31.
- these terms refer to polypeptide subunits of the above-described kinase, wherein each subunit has a corresponding molecular weight observed by SDS PAGE.
- p85 is a polypeptide subunit with approximate molecular weight of 85 KDa observed by SDS PAGE, of a large, multisubunit kinase that in its active state phosphorylates I ⁇ B ⁇ at serines 32 and 36.
- p70 has an approximate molecular weight by SDS PAGE of 70 KDa
- p62 has an approximate molecular weight by SDS PAGE of 62 kDa, and so on.
- a substrate for the kinase is a ligand which becomes phosphorylated as a result of its interaction with the kinase.
- Ligand refers to any molecule that can interact with the above- described kinase or a subunit thereof.
- the ligand can be a naturally occurring polypeptide, or may be synthetically or recombinantly produced.
- the ligand can be soluble or membrane bound.
- the ligand can also be a nonprotein molecule that acts as a ligand when it interacts with the kinase. Interactions between the ligand and the kinase include, but are not limited to, any covalent or non-covalent interactions.
- the ligand interacts selectively with the kinase.
- Agonists and antagonists of the kinase that can interact with the kinase are examples of ligands according to the present invention.
- the ligand is a selective inhibitor of the kinase activity, i.e. the ability to phosphorylate I ⁇ B- ⁇ at serine residues 32 and 36.
- disease states characterized by undesired activation of NF- ⁇ B includes, but is not limited to, disease states in a mammal which can include inflammation, HIV infection, cancer, sepsis, psoriasis, and restenosis.
- Drugs include, but are not limited to proteins, peptides, degenerate peptides, agents purified from conditioned cell medium, organic molecules, inorganic molecules, antibodies or oligonucleotides.
- Other candidate drugs include analogs ofthe above-described kinase ligand or ligands.
- the drug can be naturally occurring or synthetically or recombinantly produced.
- One skilled in the art will understand that such drugs can be developed by the assays described below. Brief Description ofthe Figures
- FIG. 1 Representation of an autoradiograph of an SDS-PAGE gel demonstrating that ubc4, but not ubc3, supports ubiquitination of I ⁇ B ⁇ .
- FIG. 2A-2E are representations of autoradiographs of SDS- PAGE gels demonstrating that the high molecular weight kinase described herein phosphorylates I ⁇ B ⁇ at serines 32 and 36.
- FIG. 3A-3E are representations of autoradiographs of SDS- PAGE gels demonstrating that under the conditions of the experiment phosphorylation of I ⁇ B ⁇ by the kinase described herein requires ubc4/ubc5.
- FIG. 4A-4B are representations of autoradiographs of SDS-
- FIG. 5A-5B are representations of autoradiographs of SDS- PAGE gels demonstrating that under the conditions of the experiment El is required for phosphorylation of I ⁇ B ⁇ by the kinase described herein. There is no requirement for okadaic acid or Rel A.
- FIG. 6A is a representation of an autoradiograph of an SDS- PAGE gel demonstrating that under the conditions ofthe experiment I ⁇ B ⁇ kinase is activated by a prior ubiqutination event. Preincubation of I ⁇ B ⁇ kinase with ubiquitination enzymes and ubiquitin eliminates the lag phase in the phosphorylation of I ⁇ B ⁇ .
- FIG. 6B is a graphical representation ofthe same data quantitated by Phosphorimager analysis.
- FIG. 7A is a representation of an autoradiograph of an SDS- PAGE gel demonstrating that I ⁇ B ⁇ kinase is ubiquitinated by ubc4.
- FIG. 7B is a graphical representation ofthe kinetics of I ⁇ B ⁇ kinase ubiqutination.
- FIG. 8 Schematic representation of the steps involved in I ⁇ B ⁇ degradation.
- ubiquitination is required both for activation of I ⁇ B ⁇ kinase and for targeting of I ⁇ B ⁇ for degradation by the proteasome.
- FIG. 9 Representation of an autoradiograph of an SDS-PAGE gel demonstrating that staurosporine and its analog K252a inhibit phosphorylation and ubiquitination of I ⁇ B ⁇ in HeLa cell extracts.
- FIG. 10A-10D are representations of autoradiographs of SDS- PAGE gels demonstrating that l ⁇ B ⁇ kinase activity is inducible and that it is activated coordinately with JNK activation.
- FIG. 11 are graphical representations of chloramphenicol acyl transferase (CAT) reporter activity, demonstrating that
- MEKK1 is required for TNF- ⁇ induction of NF- ⁇ B-dependent gene products.
- FIG. 12 Representation of an autoradiograph of an SDS-PAGE gel demonstrating that MEKKl induces site specific phosphorylation of I ⁇ B ⁇ .
- FIG. 13 A-l 3D are representations of autoradiographs of SDS- PAGE gels demonstrating that MEKK1 directly activates I ⁇ B ⁇ kinase.
- FIG. 14A-14D are representations of autoradiographs of SDS- PAGE gels in which HeLa cell cytoplasmic extract functions were assayed for I ⁇ B ⁇ kinase activity in the presence of either (A and C) ubiquitination components or (D) MEKKl ⁇ .
- FIG. 15A-15B are representations of autoradiographs of SDS- PAGE gels demonstrating that MEKKl is a selective activator of I ⁇ B ⁇ kinase.
- FIG. 16A-16B are representations of autoradiographs of SDS-
- FIG. 17 Schematic representation of a model for coordinate activation of I ⁇ B ⁇ kinase and the JNK pathway by MEKKl .
- FIG. 18 Schematic representation of a purification scheme for I ⁇ B ⁇ kinase.
- FIG. 19A is a representation of a silver stained native gel of a purified I ⁇ B ⁇ kinase fraction.
- FIG. 19B is a representation of a silver stained SDS-PAGE gel ofthe same purified I ⁇ B ⁇ kinase fraction.
- FIG. 20 Representation of an autoradiograph of an SDS-PAGE gel demonstrating inhibition of phosphorylation of 35 S-I ⁇ B ⁇ by full-length I ⁇ B ⁇ and by I ⁇ B ⁇ (5-72), an N-terminal truncation mutant of I ⁇ B ⁇ .
- FIG. 21 are amino acid sequences for pepl-pep4.
- FIG. 22 are nucleic acid sequences encoding p50 and p40, respectively. Det ailed Description of the Preferred Embodiments
- the present invention relates to a purified kinase which, in its activated state, is capable of site-specific phosphorylation of I ⁇ B ⁇ ; subunits thereof; and functional derivatives thereof. More specifically, the kinase is capable of activation by ubiquitination or via phosphorylation by MEKKl .
- the purified kinase comprise an amino acid sequence which is at least 60-100% homologous to any one ofthe amino acid sequences of Figures 21 A-2 ID or is encoded by a nucleic acid which hybridizes to a second nucleic acid having the nucleotide sequence of any one of Figures 22A-B.
- the kinase comprises an amino acid sequence that is at least 60, 65,
- the kinase comprises an amino acid sequence that is at least 90, 95, 98 or 100% homologous to any one ofthe amino acid sequences of Figures 21A-21D or is encoded by a nucleic acid which hybridizes under moderate or high stringency condition to a second nucleic acid having the nucleotide sequence of any one of Figures 22 A-B.
- the purified kinase comprises any one of the amino acid sequences of Figures 21A-21D or is encoded by a nucleic acid comprising the nucleotide sequence of any one of Figures 22A-B. More preferably, the purified kinase comprises at least 2 or 3, most preferably 4, ofthe amino acid sequences of Figures 21A-21D or amino acid sequences that are 60, 65, 70, 75, 80, or 85% homologous to the amino acid sequences of Figures 21A-21D. In a further embodiment, the present invention relates to a purified polypeptide subunit ofthe kinase complex which phosphorylates I ⁇ B ⁇ at serine residues 32 and 36 or a functional derivative thereof.
- the subunit may be a catalytic, regulatory, or structural subunit.
- the subunit is a regulatory or catalytic subunit, more preferably a catalytic subunit which when active is capable of phosphorylating I ⁇ B ⁇ .
- the subunit is selected from the group consisting of p85, p70, p62, p55, p50, p43, p40, p38, p36, p33, p31 or is a mutant or species variation thereof, or has at least 70% identity or at least 85% similarity thereto (preferably, at least 90%, 95%, 96%, 97%, 98%, or 99% identity or at least 95%, 96%, 97%, 98%, or 99% similarity thereto), or at least
- the p40 subunit or functional derivative thereof comprises an amino acid sequence that is at least 60-100% (more preferably at least 60, 65, 70, 75, 80 or 85%, most preferably at least 90, 95, 98 or 100%) homologous to the amino acid sequence of Figure 2 ID.
- the p50 subunit or functional derivative thereof comprises an amino acid sequence that is at least 60-100% (more preferably at least 60, 65, 70, 75, 80 or 85%, more preferably at least 90, 95, 98 or 100%) homologous to any one of, or any combinantion of, the amino acid sequences of Figures 21A-21C.
- the invention relates to an epitope of the above-described subunit polypeptide.
- the epitope of these polypeptides is an immunogenic or antigenic epitope.
- An immunogenic epitope is that part of the protein which elicits an antibody response when the whole protein is the immunogen.
- An antigenic epitope is a fragment of the protein which can elicit an antibody response. Methods of selecting antigenic epitope fragments are well known in the art. See, Sutcliffe et al, Science 279:660-666 (1983).
- Antigenic epitope-bearing peptides and polypeptides ofthe invention are useful to raise an immune response that specifically recognizes the polypeptides.
- Antigenic epitope-bearing peptides and polypeptides of the invention comprise at least 7 amino acids (preferably, 9, 10, 12, 15 or 20 amino acids) of the proteins of the invention.
- the kinase activity described here has several properties expected for a bona fide I ⁇ B ⁇ kinase.
- the activated kinase phosphorylates both free I ⁇ B ⁇ and I ⁇ B ⁇ bound to RelA at serine residues 32 and 36.
- I ⁇ B ⁇ phosphorylated by this kinase remains bound to NF- ⁇ B, a property expected from in vivo studies (reviewed by Finco & Baldwin, Immunity 3:263-272 (1995)).
- I ⁇ B ⁇ can be ubiquitinated at specific lysine residues (K21 and K22) (Chen, Z.J. et al, Genes & Dev. 9: 1586-1597 (1995); Scherer et al., Proc. Natl Acad. Sci. USA 92. 11259-1 1263 (1995); Baldi et al, J. Biol. Chem. 271:376-379 (1996)), and these residues are required for signal-induced degradation of I ⁇ B ⁇ in vivo
- the phosphatase inhibitor, okadaic acid was previously demonstrated to induce the phosphorylation and ubiquitination of in vitro translated I ⁇ B ⁇ in a HeLa cell cytoplasmic extract (Chen, Z.J. et al, Genes & Dev. 9: 1586-1597 (1 95)).
- the cytoplasmic extracts were separated into two fractions by monoQ ion- exchange chromatography. Fraction I (Fr.I) is the monoQ column flow-through, while fraction II (Fr.II) is the 0.5 M KCl eluate.
- the reaction mixure contained in vitro translated 5 S-labeled I ⁇ B ⁇ , an ATP regenerating system, El, ubiquitin, ubiquitin aldehyde (to inhibit isopeptidases) and okadaic acid (Chen, Z.J. et al. Genes & Dev. 9: 1586-1597 (1995)).
- Fraction II was subjected to further purification as a means of identifying the Ii B ⁇ kinase and E3.
- the observation that signal- induced phosphorylation of 35 S-labeled I ⁇ B ⁇ leads to a reduction in its electrophoretic mobility on SDS-PAGE was taken advantage of (Chen, Z.J. et al, Genes & Dev. 9:1586-1597 (1995)).
- Fraction II was separated by FPLC/monoQ chromatography into five fractions using, isocratic elution with 0.1M, 0.2M, 0.3M, 0.4M and 0.5M KCl. The I ⁇ B ⁇ kinase activity was detected in the 0.3M fraction.
- the peak of kinase activity again eluted in fraction 19.
- Ovalbumin. which was included as a carrier protein, eluted in a fraction corresponding to its expected molecular mass (43 kDa).
- the apparent high molecular weight of the kinase is not due to the formation of non-specific aggregates.
- the kinase-containing fraction was analyzed on a native polyacrylamide gel, three predominant high molecular weight bands were observed. Although the molecular weights of these complexes cannot be accurately estimated on the native gel system, the bands migrated with mobilities corresponding to molecular weights of approximately 700 kDa, consistent with the size predicted by gel filtration.
- a critical test of the specificity of the kinase activity is to determine whether it phosphorylates serine residues 32 and 36 of I ⁇ B ⁇ .
- a panel of I ⁇ B ⁇ mutants was tested including: ⁇ N (N-terminal truncation at amino acid 72), S32A/S36A (serine to alanine substitutions at residues 32 and 36), and S32E/S36E (serine to glutamic acid substitutions at residues 32 and 36) (Brockman, J.A. et al, Mol. Cell. Biol. 75:2809-2818 (1995)).
- phosphorylation of wild type I ⁇ B ⁇ resulted in a reduction in the electrophoretic mobility (lane 2).
- I ⁇ B ⁇ that can be phosphorylated are serines 32 and 36 (Haskill, S. et al, Cell 65:1281-1289 (1991)), it was concluded that phosphorylation of I ⁇ B ⁇ by the high molecular weight I ⁇ B ⁇ kinase occurs at serines 32 and 36.
- UBC4/UBC5 may be required for the phosphorylation of I ⁇ Ba
- the I ⁇ B ⁇ phosphorylation assays were initially carried out under the same conditions as the assays for the ubiquitination of I ⁇ B ⁇ , i.e., fraction I was always added to the reaction. When fraction I was omitted from the reaction, phosphorylation of I ⁇ B ⁇ was markedly reduced ( Figure 3 A, lanes 4 and 5). As expected, fraction I did not stimulate the phosphorylation of the I ⁇ B ⁇ mutant, S32A/S36A (lanes 1 and 2). The kinase-stimulatory activity of fraction I could be replaced by purified recombinant yeast UBC4 or human UBC5 (GST-UBCh5) ( Figure 3B).
- Ubiquitin may be required for the phosphorylation ofI ⁇ Ba
- MeUb cannot be multi-ubiquitinated due to the lack of free lysine residues in MeUb (Hershko & Heller, Biochem. Biophys. Res. Comm. 725:1079-1086 (1985)).
- the UBC4 UBC5 and ubiquitin requirement for the phosphorylation of I ⁇ B ⁇ prompted us to determine whether El is also required for this activity.
- the I ⁇ B ⁇ used in these experiments was translated in a wheat germ extract which contains wheat El. Thus, it was necessary to isolate I ⁇ B ⁇ from the extract by immunoprecipitation. I ⁇ B ⁇ was first allowed to associate with recombinant RelA homodimer, and the complex was then precipitated with the antisera against RelA. The immunoprecipitates were used directly as a substrate in the I ⁇ B ⁇ phosphorylation assay. As shown in Figure 5A, phosphorylation of I ⁇ B ⁇ required the addition of El or wheat germ extract. This experiment also showed that no other component in the wheat germ extract was required for the phosphorylation of I ⁇ B ⁇ .
- the phosphatase inhibitor okadaic acid is necessary to observe phosphorylation and ubiquitination of I ⁇ B ⁇ in crude HeLa cell cytoplasmic extracts (Chen, Z.J. et al. , Genes & Dev. 9: 1586- 1597 ( 1995)). These extracts also contained substantial amounts of rel proteins. Thus, it is possible that I ⁇ B ⁇ must be in a complex with rel proteins in order to be accurately phosphorylated. For these reasons, all phosphorylation assays described above contain okadaic acid and recombinant RelA homodimers.
- the partially purified I ⁇ B ⁇ kinase was preincubated with El, UBC4 and ubiquitin in the presence of ATP at 37°C for 10 min, and then initiated the phosphorylation reaction b the addition of 35 S-labeled
- I ⁇ B ⁇ ( Figure 6A, lanes 10-13; Figure 6B).
- the preincubation eliminated the lag, phase of I ⁇ B ⁇ phosphorylation.
- the lag phase of I ⁇ B ⁇ phosphorylation persisted.
- the extent of I ⁇ B ⁇ phosphorylation was reduced slightly when I ⁇ B ⁇ kinase was included in the preincubation mixture (compare lanes 5, 9, and 13 in Figure 6A). This is probably due to the instability of I ⁇ B ⁇ kinase when incubated at 37°C for an additional 10 minutes.
- Another example of the coupling of phosphorylation and ubiquitination is provided by the ubiquitination of mitotic cyclins by the cyclosome, a 20S complex that harbors an E3 activity (E3-C; Sudakin, V. et al, Mol. Biol Cell 6:185-198 (1995); King, R.W. et al, Cell 57:279-288 (1995)).
- E3-C Sudakin, V. et al, Mol. Biol Cell 6:185-198 (1995); King, R.W. et al, Cell 57:279-288 (1995)
- E3-C is dependent on cdc2, a cyclin-dependent kinase.
- the activation of E3-C by cdc2 is indirect, however, since pretreatment of cyclosome with cdc2 abolishes a lag phase in the ubiquitination of cyclins. Therefore, a phosphorylation cascade may be propagated from cdc2 within the cyclosome. The determination of the role of this kinase in vivo will require the identification and inactivation ofthe gene(s) encoding the kinase complex.
- This covalent modification ofthe I ⁇ B ⁇ kinase complex could activate the kinase by inducing a conformational change, or ubiquitination could inactivate a kinase inhibitor.
- CDK cycl in-dependent kinase
- a ubiquitination event may be required for the activation of the I ⁇ B ⁇ kinase in vitro ( Figure 8), but it has not been determined whether this event is regulated in vivo. It is possible that the kinase activity is constitutive in vivo and I ⁇ B ⁇ phosphorylation is controlled by inactivation of a phosphatase. Alternatively, the kinase activity is regulated by signals that induce NF- ⁇ B. The former possibility is consistent with the observation that okadaic acid is not required to activate the kinase activity in our most purified fractions (Figure 5B).
- the I ⁇ B ⁇ kinase was purified from unstimulated HeLa cell extracts.
- HeLa cells may have a relatively high level of "constitutive" kinase activit ' compared to normal (untransformed) cells.
- NF- ⁇ B can be activated by okadaic acid alone in transformed cells, whereas an additional signal such as H 2 0 2 is required to activate NF- ⁇ B in primary cells (Menon, S.D. et al, J. Biol. Chem.
- ROIs reactive oxygen intermediates
- NF- ⁇ B signal transduction is that NF ⁇ KB inducers cause oxidative stress, thus enhancing the multi-ubiquitination of a component of the I ⁇ B ⁇ kinase. which leads to the phosphorylation of I ⁇ B ⁇ at specific sites.
- the oxidative target could be the kinase, the E3 or other components ofthe ubiquitination machinery. It seems unlikely, however, that E2s are the targets of ROIs.
- UBC4/UBC5 The activity of UBC4/UBC5 is generally considered to be constitutive, though the synthesis of the proteins can be up-regulated in response to stress (Seufert & Jentsch, EMBO J. 9:543-550 (1990)). Since the phosphorylation of I ⁇ B ⁇ occurs within minutes following stimulation, and it does not require new protein synthesis, it is unlikely that phosphorylation of I ⁇ B ⁇ is regulated a the level of UBC4 UBC5 synthesis. These E2s alone probably do not determine substrate specificities, since they participate in the ubiquitination of a variety of proteins. Instead, these E2s function in conjunction with specific E3s to recognize target proteins. The specific recognition of phosphorylated I ⁇ B ⁇ probably requires an E3 (E3y, Figure 8), which remains to be identified.
- an ubiquitination event may be required for the activation of an I ⁇ B ⁇ kinase, which phosphorylates I ⁇ B ⁇ at serines 32 and 36.
- This two-step ubiquitination pathway for l ⁇ B ⁇ degradation is illustrated in Figure 8.
- the ubiquitination of the I ⁇ B ⁇ kinase does not require an exogenous E3, suggesting that the I ⁇ B ⁇ kinase complex possesses an E3 activity (E3 X , Figure 8).
- the activated kinase then phosphorylates I ⁇ B ⁇ , thus targeting the inhibitor to the ubiquitin proteasome pathway.
- the E3 required for the second ubiquitination event (E3 y ) may be distinct from E3 X since the partially purified kinase is not capable of ubiquitinating I ⁇ B ⁇ in the presence of El, UBC4/UBC5 and Ub.
- Casein kinase II and a 42 kDa kinase (probably similar to casein kinase II) binds to I ⁇ B ⁇ and catalyzes the basal phosphorylation of I ⁇ B ⁇ in vitro.
- deletion of a C- terminal region of I ⁇ B ⁇ that includes the PEST sequence, or mutation of the basal phosphorylation sites does not prevent the inducible phosphorylation of I ⁇ B ⁇ (Brown, K. et al, Science 267:1485-1491 (1995); Brockman, J.A. et al, Mol Cell Biol 75:2809-2818 (1995); Traenckner, E.B.-M. et al, EMBO J.
- okadaic acid is employed strictly as a phosphatase inhibitor (i.e., to preserve the phosphorylated I ⁇ B ⁇ species) rather than as an inducer of I ⁇ B ⁇ phosphorylation (Thevenin, C et al, New Biol 2:793-800 (1990); Traenckner, E.B.-M.
- I ⁇ B ⁇ kinase activity is essentially absent at 60 min (FIG. 10B, lane 8). It is formally possible that the I ⁇ B ⁇ kinase activity is constitutive and that TNF- ⁇ treatment simply results in the inactivation of a phosphatase in the extract that dephosphorylates I ⁇ B ⁇ . To address this possibility, the same extract were incubated with 35 S-labeled I ⁇ B ⁇ in the absence of okadaic acid (FIG. 10B, lanes 1, 3, 5, and 7). Under these conditions, the I ⁇ B ⁇ mobility shift is completely abolished (for example, compare lanes 3 and 4).
- TNF- ⁇ treatment cannot be accounted for solely by inactivation of an okadaic acid-sensitive I ⁇ B ⁇ phosphatase, implying that TNF- ⁇ treatment induces I ⁇ B ⁇ kinase activity.
- TNF- ⁇ treatment induces I ⁇ B ⁇ kinase activity.
- the rapid induction of I ⁇ B ⁇ kinase activity correlates with the rapid appearance of the phosphorylated form of I ⁇ B ⁇ .
- TNF- ⁇ treatment also leads to the activation of c-Jun by JNK (Hibi, M. et al, Genes & Dev. 7:2135-2148 (1993)).
- JNK Hibi, M. et al, Genes & Dev. 7:2135-2148 (1993)
- I ⁇ B ⁇ kinase activity is readily detected in S100 cytoplasmic extracts prepared from unstimulated HeLa cells using the hypotonic lysis procedure (Chen, Z.J. et al, Genes & Dev. 9:1586-1597 (1995)). It is possible that stress pathways are activated by this procedure, since another form of osmotic stress, hyperosmolar shock, has been shown to be an efficient activator ofthe JNK pathway (Galcheva-Gargova, Z. et al, Science 265:806-808 (1994)). Indeed, both the I ⁇ B ⁇ kinase and the JNK activities were detected when the S 100 extracts were incubated for 60 min (FIG. 10D). A time-dependent activation of
- JNK was detected when the S 100 extracts were incubated and then examined by an in-gel kinase assay employing the JNK substrate ATF-2.
- both the JNK and I ⁇ B ⁇ kinase may be activated during incubation of the S100 extracts, possibly owing to the hypotonic lysis conditions.
- the IFN- ⁇ enhancer contains multiple positive regulatory domains (PRDs) that bind distinct transcription factors, including NF- ⁇ B (PRDII) and AFT-2/c-Jun (PRDV) (reviewed in Thanos, D. et al. Cold Spring Harbor Sump. Quant. Biol. 55:73-81
- HeLa cells were transfected with reporters linked to either two copies of PRDII (PII), six copies of PRDIV (PIV), or the intact IFN- ⁇ enhancer (IFN), which includes these as well as other PRDs, and either an expression vector for MEKKl or an expression vector alone.
- PII PRDII
- PIV PRDIV
- IFN IFN- ⁇ enhancer
- MEKKl and MEKKl ⁇ refer to the 672 and 321 residue C-terminal fragments, respectively, ofthe full-length molecule (for discussion, see Xu, S. et al, Proc. Natl. Acad. Sci. (USA) 93:5291-5295 (1996)). Both kinases are constitutively active and indistinguishable in transfection studies.
- MEKKl activates the reporter linked to a multitimer of PRDIV (FIG. 1 1 A), which binds to either an AFT-2 homodimer or an AFT-2/c-Jun heterodimer (Du, W. and Maniatis, T.. Cell 74:887-898 (1993)).
- AFT-2 and c-Jun contain transcriptional activation domains that are phosphorylated by the JNK pathway
- MEKKl also activates the PRDII reporter.
- MEKKl does not activate all promoters, since its effect on a reporter gene containing the intact IFN- ⁇ enhancer is only marginal. This enhancer contains additional PRDs that bind factors other than NF- ⁇ B or ATF-2/c-June (see Thanos, D. et al. Cold Spring Harbor Sump. Quant. Biol.
- the IFN- ⁇ enhancer is effectively activated by virus infection, which activates all ofthe PRDs.
- MEKKl can activate both ATF-2/c-Jun and NF- ⁇ B in vivo.
- HeLa cells were trasnfected with expression vectors for Flag- tagged wild-type or mutant (S32A/S36A) I ⁇ B ⁇ , and an expression vector for MEKKl or the expression vector alone. I ⁇ B ⁇ was then immunoprecipitated with anti-Flag antibodies, and then visualized by Western blotting using anti-I ⁇ B ⁇ antibodies. MEKKl induces the appearance of an I ⁇ B ⁇ species with reduced mobility compared to that isolated from uninduced cells ( Figure 12A, land 2).
- MEKKl ⁇ alone fails to induce this site-specific phosphorylation (lane 1).
- extracts were incubated with MEKKl ⁇ in the absence of presence of the phosphatase inhibitor okadaic acid ( Figure 13B).
- the MEKKl ⁇ -induced I ⁇ B ⁇ shift is largely abolished (compare lanes 2 and 3).
- the effects of MEKKl ⁇ cannot be accounted for solely by the inactivation of an okadaic acid-sensitive I ⁇ B ⁇ phosphatase, implying that MEKKl ⁇ activates the I ⁇ B ⁇ kinase.
- MEKK ⁇ coordinately activates the I ⁇ B ⁇ kinase and JNK pathways in cytoplasmic extracts.
- I ⁇ B ⁇ could be a substrate for MEKKl , MKK4, or JNK.
- MKK4 nor JNK1 phosphorylated I ⁇ B ⁇ , with appropriate control experiments demonstrating that these proteins were enzymatically active.
- MEKKl ⁇ did phosphorylate I ⁇ B ⁇ directly; however, the degree of phosphorylation was over 10-fold less than that seen with MKK4 as a substrate, and. as shown below, MEKK1 ⁇ does not phosphorylate I ⁇ B ⁇ at Ser-32 or -36.
- I ⁇ B ⁇ kinase activity resides in a large, approximately 700 kDa, multiprotein complex (Chen, Z.J. el al, Cell
- I ⁇ B ⁇ immunoprecipitation I ⁇ B ⁇ was employed as a substrate. As shown in lane 7, this I ⁇ B ⁇ is also a substrate for MEKKl ⁇ -activated I ⁇ B ⁇ kinase. Thus, MEKKl ⁇ activation of the I ⁇ B ⁇ kinase is direct. Additional experiments indicate that MEKKl ⁇ is a potent activator of the I ⁇ B ⁇ kinase ( Figure 13D), with activation demonstratible with MEKKl ⁇ doses as low as 5 ng (lane 2).
- I ⁇ B ⁇ complexed with RelA is a substrate for MEKKl ⁇ -activated I ⁇ B ⁇ kinase, just as it is for the ubiquitination-activated kinase.
- the MEKKl -Inducible I ⁇ Ba Kinase Is a High Molecular Weight Complex
- the peak of MEKKl ⁇ -inducible I ⁇ B ⁇ kinase activity elutes at a position (fractions 19 to 20) corresponding to a native molecular weight of approximately 700 kDa, indistinguishable from that of the ubiquitination-inducible I ⁇ B ⁇ kinase (Chen, Z.J. et al, Cell 54:853-862 ( 1996)).
- Further fractionation by anion exchange chromatography reveals that the MEKKl ⁇ -inducible I ⁇ B ⁇ kinase activity elutes in a broader peak than the ubiquitination-inducible activity ( Figures 14C and 14D).
- JNK raises the possibility that potential upstream activators of MEKK 1 , such as the small GTP-binding proteins Racl, Cdc42, and Ras, as well as protein kinases that they activate, such as PAK, may also be common elements of a single upstream signal transduction mechanism.
- potential upstream activators of MEKK 1 such as the small GTP-binding proteins Racl, Cdc42, and Ras, as well as protein kinases that they activate, such as PAK
- PKA has been shown to dissociate the NF- ⁇ B-I ⁇ B complex (Ghosh, S. and Baltimore, D., Nature 344:678-682 (1990)), while PKC ⁇ coimmunoprecipitates with a factor that can phosphorylate I ⁇ B ⁇ (Diaz-Meco, M.T. et al, EMBO J. 73:2842-2848 (1994)); with regard to the latter, it has been suggested that PKC ⁇ activates a kinase that
- I ⁇ B ⁇ Neither PKA nor PKC ⁇ , however, phosphyroylates I ⁇ B ⁇ at Ser-32 and - 36, nor does either activate the I ⁇ B ⁇ kinase ( Figure 15 A). Additional kinases that have been implicated in NF- ⁇ B activation are raf- 1 and the double-stranded RNA-activated protein kinase (PKR) (Finco, T.S. and Baldwin, A.S., J. Biol Chem. 265:17676-17679 (1993); Yang, Y.L. et al, EMBO J. 74:6095-6106
- MEKKl is a member of a family of enzymes that share a conserved C- terminal catalytic domain and may thus share overlap in substrates (Lange-Carter, CA. et al. Science 260:315-319 (1993); Blank, J.L. et al, J. Biol Chem.
- MEKK isoforms other than MEKKl can activate the I ⁇ B ⁇ kinase.
- Different MEKK isoforms could potentially be involved in signaling responses to different stimuli.
- MEKKl has been shown to bind Ras in a GTP-dependent manner and thus its activity may be regulated in a similar fashion (Russell, M. et al, J. Biol. Chem. 270: ⁇ ⁇ 757- 11760 (1995)).
- MEKKs could conceivably be regulated by other upstream regulators such as Racl, Cdc42, and PAK. These data also leave open the possibility that there may be other I ⁇ B ⁇ kinases that respond to stimuli distinct from those that signal through MEKKl .
- I ⁇ B ⁇ kinase and MEKKl are indeed part ofthe same pathway.
- the I ⁇ B ⁇ kinase can be activated by ubiquitination independently of phosphorylation ( Figure 17). This dual regulation by phosphorylation or ubiquitination is unprecedented. Thus, the I ⁇ B ⁇ kinase itself can be considered a signal integrator, responding to both phosphorylation and ubiquitination. Different stimuli may therefore activate one, the other, or both pathways. In principle, then, it may be possible to isolate an induced I ⁇ B ⁇ kinase species that is not ubiquitination-dependent, or one that is not phosphorylation-dependent. Indeed, the fractionation of highly purified I ⁇ B ⁇ kinase by ion exchange chromatography reveals kinase species that are phosphorylation but not ubiquitination inducible ( Figures 14C and 14D).
- I ⁇ B ⁇ kinase from SlOO extracts removes it from both the ubiquitination components and MEKKl present in the extract; in fact, Western blotting indicates that MEKKl is not present in the I ⁇ B ⁇ kinase complex. Either ubiquitination or MEKKl -dependent phosphorylation can activate the purified I ⁇ B ⁇ kinase ( Figure 13C).
- MEKKl activates the I ⁇ B ⁇ kinase.
- MEKKl inactivates a negative regulatory subunit of the I ⁇ B ⁇ kinase, just as cAMP binds to and induces the dissociation of the regulatory subunit of PKA (Francis. S.H. and Corbin, J.O.,Annu. Rev. Physiol 56:237-272 (1994)).
- MEKKl may activate the catalytic subunit ofthe I ⁇ B ⁇ kinase that subsequently phosphorylates Ser-32 and -36 of I ⁇ B ⁇ .
- MEKKl initiates a MAPK-like cascade within the I ⁇ B ⁇ kinase complex, with the terminal kinase the subunit that phosphorylates Ser-32 and -36; this would be somewhat analogous to the organization of MAPK modules as high molecular weight complexes in yeast (Choi, K.Y. et al, CeU 75:499-512 (1994)).
- the incorporation of 32 P into multiple subunits of the I ⁇ B ⁇ kinase complex in the presence of MEKKl ⁇ ( Figure 16) could be consistent with any of these possibilities.
- Amino acid sequence variants of the above-described kinase. subunits thereof or functional derivatives thereof can be prepared by mutations in the DNA or by chemical synthesis. Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence of the kinase subunits or derivatives. Any combination of deletion, insertion, and substitution can also be made to arrive at the final construct, provided that the final construct possesses the desired activity. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure.
- the mutation er se need not be predetermined.
- random mutagenesis can be conducted at the target codon or region and the expressed above-described kinase subunit variants screened for the optimal combination of desired activity.
- Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, site-specific mutagenesis.
- Preparation of an above-described kinase variant in accordance herewith is preferably achieved by site-specific mutagenesis of DNA that encodes an earlier prepared variant or a nonvariant version of the protein.
- Site-specific mutagenesis allows the production of kinase variants through the use of specific oligonucleotide sequences that encode the DNA sequence ofthe desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides ofthe deletion junction being traversed.
- a primer of about 20 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction ofthe sequence being altered.
- the technique of site-specific mutagenesis is well known in the art. as exemplified by publications such as Adelman et al. , DNA 2: 183 (1983).
- the site-specific mutagenesis technique typically employs a phage vector that exists in both a single-stranded and double-stranded form.
- Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage, for example, as disclosed by Messing et al. Third Cleveland Symposium on Macromolecules and Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981). These phage are readily commercially available and their use is generally well known to those skilled in the art.
- plasmid vectors that contain a single-stranded phage origin of replication (Vieira et al, Meth. Enzymol. 153:3 (1987)) can be employed to obtain single-stranded
- site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector that includes within its sequence a DNA sequence that encodes the relevant protein.
- An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example, by the method of Crea et al, Proc. Natl Acad. Sci. (USA) 75:5765 (1978).
- This primer is then annealed with the single-stranded protein-sequence- containing vector, and subjected to DNA-polymerizing enzymes such as E. coli polymerase I Klenow fragment, to complete the synthesis ofthe mutation-bearing strand.
- DNA-polymerizing enzymes such as E. coli polymerase I Klenow fragment
- This heteroduplex vector is then used to transform appropriate cells and clones are selected that include recombinant vectors bearing the mutated sequence arrangement. After such a clone is selected, the mutated protein region can be removed and placed in an appropriate vector for protein production, generally an expression vector of the type that can be employed for transformation of an appropriate host.
- Amino acid sequence deletions generally range from about 1 to 30 residues, more preferably 1 to 10 residues, and typically are contiguous.
- Amino acid sequence insertions include amino and/or carboxyl-terminal fusions of from one residue to polypeptides of essentially unrestricted length, as well as intrasequence insertions of single or multiple amino acid residues.
- Intrasequence insertions i.e., insertions within a complete kinase subunit sequence
- the third group of variants are those in which at least one amino acid residue in the above-described kinase molecule, and preferably, only one, has been removed and a different residue inserted in its place. Such substitutions preferably are made in accordance with the following Table 1 when it is desired to modulate finely the characteristics of the above-described kinase.
- Substantial changes in functional or immunological identity are made by selecting substitutions that are less conservative than those in Table 1, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk ofthe side chain.
- substitutions that in general are expected to those in which (a) glycine and/or proline is substituted by another amino acid or is deleted or inserted; (b) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (c) a cysteine residue is substituted for (or by) any other residue; (d) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyi, is substituted for (or by) a residue having an electronegative charge, e.g., glutamyl or aspartyl; or (e) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having such a side chain, e
- a variant typically is made by site-specific mutagenesis of a native kinase subunit encoding-nucleic acid, expression ofthe variant nucleic acid in recombinant cell culture, and, optionally, purification from the cell culture, for example, by immunoaffmity adso ⁇ tion on a column (to absorb the variant by binding it to at least one remaining immune epitope).
- the activity ofthe cell lysate or purified kinase molecule variant is then screened in a suitable screening assay for the desired characteristic.
- a change in the immunological character of the above-described kinase molecule, such as affinity for a given antibody is measured by a competitive type immunoassay. Changes in immunomodulation activity are measured by the appropriate assay. Modifications of such protein properties as redox or thermal stability, hydrophobicity, susceptibility to proteolytic degradation or the tendency to aggregate with carriers or into multimers are assayed by methods well known to the ordinarily skilled artisan.
- the peptide or polypeptide ofthe present invention is purified from tissues or cells which naturally produce the peptide.
- the above-described isolated nucleic acid fragments could be used to express the above-described kinase or subunit thereof in any organism.
- the samples ofthe present invention include cells, protein extracts or membrane extracts of cells, or biological fluids. The sample will vary based on the assay format, the detection method and the nature ofthe tissues, cells or extracts used as the sample.
- the peptide or polypeptide can be chemically synthesized, for example, using an automated solid-phase peptide synthesizer (See, Ausubel, F.M. et al, Current protocols in Molecular Biology, John Wiley & Sons, Inc., New York, NY (1989), specifically, at page 18.6.1 1 of Supplement 35).
- source organism refers to the original organism from which the amino acid sequence of the subunit is derived, regardless of the organism the subunit is expressed in and ultimately isolated from.
- the purification procedures comprise ion- exchange chromatography and size exclusion chromatography.
- ion-exchange resins known in the art can be employed, including for example, monoQ, sepharose Q, macro-prepQ, AG1-X2, or HQ.
- suitable size exclusion resins include, but are not limited to, Superdex 200,
- the present invention relates to an isolated nucleic acid molecule coding for a polypeptide having an amino acid sequence corresponding to the above-described kinase or to a subunit of the above- described kinase.
- the isolated nucleic acid molecule which encodes a polypeptide having an amino acid sequence corresponding to the above-described kinase hybridizes to a second nucleic acid molecule having the nucleotide sequence set forth in any one of Figures 22A-B. More preferably, said isolated nucleic acid hybridizes preferentially or hybridizes under low stringency conditions, even more preferably moderate stringent, and even more preferably under high stringency conditions to a second nucleic acid having the nucleotide sequence of any one of Figures 22A-B. Most preferably, the nucleic acid molecule comprises the nucleotide sequence set forth in any one of Figures 22 A-B.
- nucleic acid sequences can be altered by substitutions, additions or deletions that provide for functionally equivalent molecules.
- nucleic acid sequences can be altered by substitutions, additions or deletions that provide for functionally equivalent molecules.
- nucleotide coding sequences other DNA sequences which encode substantially the same amino acid sequence as a kinase subunit can be used in the practice ofthe present invention. These include but are not limited to nucleotide sequences comprising all or portions of the kinase subunit nucleic acids which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
- nucleic acid sequence affords an opportunity to promote secretion and/or processing of heterologous proteins encoded by foreign nucleic acid sequences fused thereto.
- All variations of the nucleotide sequence of the above-described kinase gene and fragments thereof permitted by the genetic code are, therefore, included in this invention.
- the nucleic acid sequence can comprise a nucleotide sequence which results from the addition, deletion or substitution of at least one nucleotide to the 5'-end and/or the 3'-end ofthe nucleic acid formula ofthe above-described kinase gene or a derivative thereof.
- Any nucleotide or polynucleotide can be used in this regard, provided that its addition, deletion or substitution does not alter the amino acid sequence ofthe above-described kinase gene which is encoded by the nucleotide sequence.
- the nucleic acid molecule of the present invention can, as necessary, have restriction endonuclease recognition sites added to its 5 '-end and/or 3 '-end.
- the two polypeptides are functionally equivalent, as are the two nucleic acid molecules which give rise to their production, even though the differences between the nucleic acid molecules are not related to degeneracy of the genetic code.
- nucleic acid molecules coding for polypeptides having amino acid sequences corresponding to the above- described kinase subunits are provided.
- the nucleic acid molecule can be isolated from a biological sample containing human RNA or DNA.
- the nucleic acid molecule can be isolated from a biological sample containing human RNA using the techniques of cDNA cloning and subtractive hybridization.
- the nucleic acid molecule can also be isolated from a cDNA library using a homologous probe.
- the nucleic acid molecule can be isolated from a biological sample containing human genomic DNA or from a genomic library. Suitable biological samples include, but are not limited to, blood, semen and tissue. The method of obtaining the biological sample will vary depending upon the nature of the sample.
- the isolated nucleic acid molecule is also intended to include allelic variations, so long as the sequence is a functional derivative ofthe above-described kinase gene.
- the kinase allele does not encode the identical sequence to a known allele. it can be isolated and identified as the kinase allele using the same techniques used herein, and especially PCR techniques to amplify the appropriate gene with primers.
- kinase subunit genes for example, eukaryotes; more specifically, mammals, birds, fish, and plants; more specifically, gorillas, rhesus monkeys, and chimpanzees.
- the invention is intended to include, but not be limited to, above- described kinase subunit nucleic acid molecules isolated from the above- described organisms.
- Isolated nucleic acid molecules ofthe present invention are also meant to include those chemically synthesized.
- a nucleic acid molecule with the nucleotide sequence which codes for the expression product of the above- described kinase gene can be designed and, if necessary, divided into appropriate smaller fragments. Then an oligomer which corresponds to the nucleic acid molecule, or to each ofthe divided fragments, can be synthesized.
- Such synthetic oligonucleotides can be prepared, for example, by the triester method of Matteucci et al, J. Am. Chem. Soc. 703:3185-3191 (1981) or by using an automated DNA synthesizer.
- An oligonucleotide can be derived synthetically or by cloning. If necessary, the 5 '-ends of the oligomers can be phosphorylated using T4 polynucleotide kinase. Phosphorylation of single strands prior to annealing or for labeling can be achieved using an excess ofthe enzyme. If phosphorylation is for the labeling of probe, the ATP can contain high specific activity radioisotopes. Then, the DNA oligomer can be subjected to annealing and ligation with T4 ligase or the like.
- the present invention relates to a nucleic acid probe for the specific detection ofthe presence of the above-described kinase or a subunit thereof in a sample comprising the above-described nucleic acid molecules or at least a fragment thereof which binds under stringent conditions to a nucleic acid molecule encoding the above-described kinase or a subunit thereof.
- the present invention relates to an isolated nucleic acid probe consisting of 10 to 1000 nucleotides (prefererably, 10 to 500, 10 to 100, 10 to 50, 10 to 35, 20 to 1000, 20 to 500, 20 to 100, 20 to 50, or 20 to 35) which hybridizes preferentially to RNA or DNA of the above-described kinase or a subunit thereof (preferably, the probe will hybridize only to sequences which fully or partially encode the above-described kinase or subunit thereof), wherein said nucleic acid probe is or is complementary to a nucleotide sequence consisting of at least 10 consecutive nucleotides (preferably, 15, 20, 25. or 30) from the nucleic acid molecule comprising a polynucleotide sequence at least 90% identical to a sequence of the above-described kinase or a subunit thereof.
- nucleic acid probe consisting of 10 to 1000 nucleotides (prefererably, 10 to 500, 10 to 100, 10 to 50, 10
- the nucleic acid probe can be used to probe an appropriate chromosomal or cDNA library by usual hybridization methods to obtain another nucleic acid molecule of the present invention.
- a chromosomal DNA or cDNA library can be prepared from appropriate cells according to recognized methods in the art (cf. Molecular Cloning: A Laboratory Manual, second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, 1989).
- nucleic acid probes having nucleotide sequences which correspond to N-terminal and C-terminal portions of the amino acid sequence of the Above-described kinase or subunit thereof.
- the synthesized nucleic acid probes can be used as primers in a polymerase chain reaction (PCR) carried out in accordance with recognized PCR techniques, essentially according to PCR Protocols, A Guide to Methods and Applications, edited by Michael et al, Academic Press, 1990, utilizing the appropriate chromosomal or cDNA library to obtain the fragment of the present invention.
- PCR polymerase chain reaction
- hybridization probes of the present invention can be labeled by standard labeling techniques such as with a radiolabel, enzyme label, fluorescent label, biotin-avidin label, chemiluminescence, and the like. After hybridization, the probes can be visualized using known methods.
- the nucleic acid probes ofthe present invention include RNA, as well as DNA probes, such probes being generated using techniques known in the art.
- a nucleic acid probe is immobilized on a solid support.
- solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins, such as polyacrylamide and latex beads. Techniques for coupling nucleic acid probes to such solid supports are well known in the art.
- the test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids. The sample used in the above-described methods will vary based on the assay format, the detection method and the nature ofthe tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample which is compatible with the method utilized.
- the present invention relates to a method of detecting the presence of the above-described kinase or subunit thereof in a sample comprising a) contacting the sample with (i) the above-described nucleic acid probe, under conditions such that hybridization occurs; (ii) a nucleic acid molecule which hybridizes to a second nucleic acid molecule having the nucleotide sequence set forth in anyone of Figures 22 A-B; or (iii) a nucleic acid molecule which comprises the nucleotide sequence set forth in anyone of Figures 22A-B and b) detecting the presence of the probe bound to the nucleic acid molecule.
- Samples to be tested include but should not be limited to RNA samples of human tissue.
- Altered expression levels of kinase subunit RNA in an individual, as compared to normal levels, can indicate the presence of disease.
- the above- described kinase probes can further be used to assay cellular activity in general.
- the present invention relates to a kit for detecting the presence of the above-described kinase or a subunit thereof in a sample comprising at least one container means having disposed therein the above- described nucleic acid molecule.
- the kit further comprises other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound nucleic acid probe.
- detection reagents include, but are not limited to radiolabelled probes, enzymatic labeled probes (horse radish peroxidase, alkaline phosphatase), and affinity labeled probes (biotin, avidin, or steptavidin).
- a compartmentalized kit includes any kit in which reagents are contained in separate containers.
- Such containers include small glass containers, plastic containers or strips of plastic or paper.
- Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
- Such containers will include a container which will accept the test sample, a container which contains the probe or primers used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris- buffers, and the like), and containers which contain the reagents used to detect the hybridized probe, bound antibody, amplified product, or the like.
- the present invention relates to a recombinant
- DNA molecule comprising, 5' to 3', a promoter effective to initiate transcription in a host cell and the above-described nucleic acid molecules.
- the present invention relates to a recombinant DNA molecule comprising a vector and an above-described nucleic acid molecule.
- the present invention relates to a nucleic acid molecule comprising a transcriptional control region functional in a cell, a sequence complimentary to an RNA sequence encoding an amino acid sequence corresponding to the above-described polypeptide, and a transcriptional termination region functional in the cell.
- the above-described molecules are isolated and/or purified DNA molecules.
- the present invention relates to a cell or non ⁇ human organism that contains an above-described nucleic acid molecule.
- the peptide is purified from cells which have been altered to express the peptide.
- a cell is said to be "altered to express a desired peptide" when the cell, through genetic manipulation, is made to produce a protein which it normally does not produce or which the cell normally produces at low levels.
- One skilled in the art can readily adapt procedures for introducing and expressing either genomic. cDNA, or synthetic sequences into either eukaryotic or prokaryotic cells.
- a nucleic acid molecule, such as DNA is said to be “capable of expressing” a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide.
- An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene sequence expression.
- the precise nature ofthe regulatory regions needed for gene sequence expression can vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation.
- Such regions will normally include those 5 '-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
- the non-coding region 3 ' to the sequence encoding the above- described kinase subunit gene can be obtained by the above-described methods.
- This region can be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation.
- the transcriptional termination signals can be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell can be substituted.
- Two DNA sequences are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of an above-described kinase subunit gene sequence, or (3) interfere with the ability of the above-described kinase subunit gene sequence to be transcribed by the promoter region sequence.
- a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
- the present invention encompasses the expression of any of the above- described kinase subunit genes (or a functional derivative thereof) in either prokaryotic or eukaryotic cells.
- Prokaryotic hosts are, generally, the most efficient and convenient for the production of recombinant proteins and, therefore, are preferred for the expression of the above-described kinase subunit gene.
- Prokaryotes most frequently are represented by various strains of E. coli. However, other microbial strains can also be used, including other bacterial strains.
- plasmid vectors that contain replication sites and control sequences derived from a species compatible with the host can be used. Examples of suitable plasmid vectors include pBR322, pCDNA, pGEX, pGEM, pUCl 18, pUCl 19 and the like; suitable phage or bacteriophage vectors include ⁇ gtlO, ⁇ gtl 1 and the like; and suitable virus vectors include pMAM-neo, pKRC and the like.
- the selected vector of the present invention has the capacity to replicate in the selected host cell.
- prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However, under such conditions, the peptide will not be glycosylated.
- the prokaryotic host must be compatible with the replicon and control sequences in the expression plasmid.
- kinase subunit in a prokaryotic cell, it is necessary to operably link the kinase sequence to a functional prokaryotic promoter.
- promoters can be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible).
- constitutive promoters include the int promoter of bacteriophage ⁇ , the bla promoter of the ⁇ -lactamase gene sequence of pBR322, and the CAT promoter ofthe chloramphenicol acetyl transferase gene sequence of pBR325, and the like.
- inducible prokaryotic promoters examples include the major right and left promoters of bacteriophage ⁇ (P L and P R ), the trp, recA, lacZ, lad, and gal promoters of E. coli, the ⁇ -amylase (Ulmanen et al, J. Bacteriol 762:176-182 (1985)) and the ⁇ -28-specific promoters of B.
- subtilis (Gilman et al, Gene sequence 32:1 1-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, In: The Molecular Biology ofthe Bacilli, Academic Press, Inc., NY (1982)), and Strepto ⁇ myces promoters (Ward et al, Mol Gen. Genet. 203:468-478 (1986)).
- Prokaryotic promoters are reviewed by Glick (J. Ind. Microbiol. 7:277-282 (1987)); Cenatiempo (Biochimie 65:505-516 (1986)); and Gottesman (Ann. Rev. Genet. 75:415-442 (1984)).
- ribosome binding sites are disclosed, for example, by Gold et al. (Ann. Rev.
- control sequences are dependent on the type of host cell used to express the gene.
- “cell”, “cell line”, and “cell culture” can be used interchangeably and all such designations include progeny.
- transformants or “transformed cells” include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that all progeny can not be precisely identical in DNA content, due to deliberate or inadvertent mutations. However, as defined, mutant progeny have the same functionality as that ofthe originally transformed cell.
- Host cells which can be used in the expression systems of the present invention are not strictly limited, provided that they are suitable for use in the expression of an above-described kinase subunit of interest.
- Suitable hosts include eukaryotic cells.
- Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture.
- Preferred mammalian cells include HeLa cells, cells of fibroblast origin such as VERO or CHO-Kl, or cells of lymphoid origin and their derivatives.
- plant cells are also available as hosts, and control sequences compatible with plant cells are available, such as the cauliflower mosaic virus 35S and 19S, and nopaline synthase promoter and polyadenylation signal sequences.
- Another preferred host is an insect cell, for example Drosophila larvae.
- insect cells for example Drosophila larvae.
- the Drosophila alcohol dehydrogenase promoter can be used. Rubin, Science 240:1453-1459 (1988).
- baculovirus vectors can be engineered to express large amounts ofthe above-described kinase or subunit thereof in insects cells (Jasny, Science 235:1653 (1987); Miller et al, In: Genetic Engineering (1986), Setlow, J.K., et al, eds., Plenum, Vol. 8, pp. 277-297).
- Different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, cleavage) of proteins.
- Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing ofthe foreign protein expressed.
- expression in a bacterial system can be used to produce an unglycosylated core protein product.
- Expression in yeast will produce a glycosylated product.
- Expression in mammalian cells can be used to ensure
- Any of a series of yeast gene sequence expression systems can be utilized which inco ⁇ orate promoter and termination elements from the actively expressed gene sequences coding for glycolytic enzymes are produced in large quantities when yeast are grown in mediums rich in glucose.
- Known glycolytic gene sequences can also provide very efficient transcriptional control signals.
- Yeast provides substantial advantages in that it can also carry out post- translational peptide modifications.
- Yeast recognizes leader sequences on cloned mammalian gene sequence products and secretes peptides bearing leader sequences (i.e., pre-peptides).
- leader sequences on cloned mammalian gene sequence products and secretes peptides bearing leader sequences (i.e., pre-peptides).
- pre-peptides i.e., pre-peptides
- transcriptional and translational regulatory sequences can be employed, depending upon the nature ofthe host.
- the transcriptional and translational regulatory signals can be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression.
- promoters from mammalian expression products such as actin. collagen, myosin, and the like, can be employed.
- Transcriptional initiation regulatory signals can be selected which allow for repression or activation, so that expression ofthe gene sequences can be modulated.
- regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabolite) regulation.
- Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.
- Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al, J. Mol Appl Gen. 7:273-288 (1982)); the TK promoter of He ⁇ es virus (McKnight, Cell 37:355-365 (1982)); the SV40 early promoter (Benoist et al,
- yeast gal4 gene sequence promoter Johnston et al, Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver et al, Proc. Natl Acad. Sci. (USA) 57:5951-5955 (1984)).
- kinase subunit nucleic acid molecule and an operably linked promoter can be introduced into a recipient prokaryotic or eukaryotic cell either as a non-replicating DNA (or RNA) molecule, which can either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the gene can occur through the transient expression of the introduced sequence. Alter ⁇ natively, permanent expression can occur through the integration of the intro-uted DNA sequence into the host chromosome.
- a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome.
- Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
- the marker can provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like.
- the selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements can also be needed for optimal synthesis of single chain binding protein mRNA. These elements can include splice signals, as well as transcription promoters, enhancers, and termination signals.
- cDNA expression vectors inco ⁇ orating such elements include those described by Okayama, Molec. Cell. Biol. 3:280 (1983).
- the introduced nucleic acid molecule will be inco ⁇ orated into a plasmid or viral vector capable of autonomous replication in the recipient host.
- a plasmid or viral vector capable of autonomous replication in the recipient host.
- Any of a wide variety of vectors can be employed for this pu ⁇ ose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector can be recognized and selected from those recipient cells which do not contain the vector; the number of copies ofthe vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
- Preferred prokaryotic vectors include plasmids such as those capable of replication in E.
- plasmids are, for example, disclosed by Sambrook (cf. Molecular Cloning: A Laboratory Manual second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, 1989).
- Bacillus plasmids include pC194, pC221, pT127, and the like. Such plasmids are disclosed by Gryczan (In: The Molecular Biology ofthe Bacilli, Academic Press, NY (1982), pp. 307-329).
- Suitable Streptomyces plasmids include pIJlOl (Kendall et al, J.
- Pseudomonas plasmids are reviewed by John et al (Rev. Infect. Dis. 5:693-704 (1986)), and Izaki (Jpn. J Bacteriol. 33:729-742 (1978)).
- Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40,
- the DNA construct(s) can be introduced into an appropriate host cell by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate-precipitation, direct microinjection, and the like.
- recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene molecule(s) results in the production of an above-described kinase subunit(s).
- the present invention relates to an antibody having binding affinity specifically to the above-described kinase or subunit thereof as described above or specifically to an above-described kinase binding fragment thereof.
- Those antibodies which bind selectively to the above-described kinase or a subunit thereof would be chosen for use in methods which could include, but should not be limited to, the analysis of altered kinase or subunit expression.
- the above-described kinase or subunits thereof of the present invention can be used in a variety of procedures and methods, such as for the generation of antibodies, for use in identifying pharmaceutical compositions, and for studying DNA/protein interaction.
- kinase or subunits thereof of the present invention can be used to produce antibodies or hybridomas.
- an antibody is desired, such a peptide would be generated as described herein and used as an immunogen.
- the antibodies of the present invention include monoclonal and polyclonal antibodies, as well as fragments of these antibodies.
- the invention further includes single chain antibodies.
- Antibody fragments which contain the idiotype ofthe molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab') 2 fragment; the Fab' fragments, Fab fragments, and Fv fragments.
- Humanized antibodies can be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non- immunogenic portion (i.e. chimeric antibodies) (Robinson, R.R. et al, International Patent Publication PCT/US86/02269; Akira, K. et al , European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison, S.L. et al, European Patent Application 173,494; Neuberger, M.S. et al., PCT Application WO 86/01533; Cabilly, S.
- the present invention relates to a hybridoma which produces the above-described monoclonal antibody.
- a hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.
- techniques for preparing monoclonal antibodies and hybridomas are well known in the art (Campbell, "Monoclonal Antibody
- Any animal which is known to produce antibodies can be immunized with the selected polypeptide.
- Methods for immunization are well known in the art. Such methods include subcutaneous or inte ⁇ eritoneal injection ofthe polypeptide.
- One skilled in the art will recognize that the amount of polypeptide used for immunization will vary based on the animal which is immunized, the antigenicity of the polypeptide and the site of injection.
- the polypeptide can be modified or administered in an adjuvant in order to increase the peptide antigenicity. Methods of increasing the antigenicity of a polypeptide are well known in the art.
- Such procedures include coupling the antigen with a heterologous protein (such as globulin or ⁇ -galactosidase) or through the inclusion of an adjuvant during immunization.
- a heterologous protein such as globulin or ⁇ -galactosidase
- an adjuvant during immunization.
- spleen cells from the immunized animals are removed, fused with myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells.
- any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz e/ ⁇ /., Exp. Cell Res. 75:109- 124 (1988)). Hybridomas secreting the desired antibodies are cloned and the class and subclass is determined using procedures known in the art (Campbell, Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and
- antibody containing antisera is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one ofthe above-described procedures.
- the above-described antibodies are detectably labeled.
- Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, and the like), enzymatic labels (such as horse radish peroxidase, alkaline phosphatase, and the like) fluorescent labels (such as FITC or rhodamine, and the like), paramagnetic atoms, and the like.
- the labeled antibodies of the present invention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues which express a specific peptide.
- the above-described antibodies are immobilized on a solid support.
- Such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir et al, "Handbook of Experimental Immunology” 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby et al, Meth. Enzym. 34 Academic Press, N.Y. (1974)).
- the immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as in immunochromotography.
- Anti-peptide peptides can be generated in one of two fashions.
- the anti-peptide peptides can be generated by replacing the basic amino acid residues found in the above-described kinase or subunit thereof sequence with acidic residues, while maintaining hydrophobic and uncharged polar groups. For example, lysine, arginine, and/or histidine residues are replaced with aspartic acid or glutamic acid and glutamic acid residues are replaced by lysine, arginine or histidine.
- the present invention relates to a method of detecting the above-described kinase or a subunit thereof in a sample, comprising: a) contacting the sample with an above-described antibody, under conditions such that immunocomplexes form, and b) detecting the presence ofthe antibody bound to the polypeptide.
- the methods comprise incubating a test sample with one or more of the antibodies of the present invention and assaying whether the antibody binds to the test sample. Altered levels of the above-described kinase or subunits thereof in a sample as compared to normal levels can indicate a specific disease.
- Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature ofthe antibody used in the assay.
- immunological assay formats such as radioimmunoassays, enzyme-linked immunosorbent assays, diffusion based Ouchterlony, or rocket immunofluorescent assays
- Examples of such assays can be found in Chard, An Introduction to Radioimmunoassay and Related Techniques. Elsevier Science Publishers, Amsterdam, The Netherlands
- the immunological assay test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as blood, serum, plasma, or urine.
- the test sample used in the above-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is capable with the system utilized.
- IX. A Diagnostic Kit Comprising Antibodies to the Kinase or A Subunit Thereof.
- kits which contains all the necessary reagents to carry out the previously described methods of detection.
- the kit can comprise: i) a first container means containing an above-described antibody, and ii) second container means containing a conjugate comprising a binding partner of the antibody and a label.
- the kit further comprises one or more other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound antibodies.
- detection reagents include, but are not limited to, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the chromophoric, enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.
- the compartmentalized kit can be as described above for nucleic acid probe kits.
- the antibodies described in the present invention can readily be inco ⁇ orated into one ofthe established kit formats which are well known in the art.
- the diagnostic and screening methods of the invention are especially useful for a patient suspected of being at risk for developing a disease associated with an altered expression level ofthe above-described kinase Or a subunit thereof based on family history, or a patient in which it is desired to diagnose a disease related to the above-described kinase.
- presymptomatic screening of an individual in need of such screening is now possible using DNA encoding the above-described kinase or a subunit thereof.
- the screening method of the invention allows a presymptomatic diagnosis, including prenatal diagnosis, of the presence of a missing or aberrant kinase subunit gene in individuals, and thus an opinion concerning the likelihood that such individual would develop or has developed a disease associated with the above-described kinase. This is especially valuable for the identification of carriers of altered or missing kinase genes, for example, from individuals with a family history of a disease associated with the above- described kinase. Early diagnosis is also desired to maximize appropriate timely intervention.
- a tissue sample would be taken from such individual, and screened for (1) the presence of the "normal” kinase subunit gene(s); (2) the presence ofthe kinase subunit mRNA(s) and/or (3) the presence ofthe kinase or subunits thereof.
- the normal human gene can be characterized based upon, for example, detection of restriction digestion patterns in "normal” versus the patient's DNA, including RFLP analysis, using DNA probes prepared against the kinase sequence (or a functional fragment thereof) taught in the invention.
- mRNA encoding a kinase subunit(s) can be characterized and compared to normal kinase mRNA (a) levels and/or (b) size as found in a human population not at risk of developing the kinase- associated disease using similar probes.
- the above-described kinase subunits can be (a) detected and/or (b) quantitated using a biological assay for kinase activity or using an immunological assay and the above-described kinase antibodies. When assaying the above-described kinase protein, the immunological assay is preferred for its speed.
- an (1) aberrant kinase subunit DNA size pattern, and/or (2) aberrant kinase subunit(s) mRNA sizes or levels and/or (3) aberrant kinase protein levels would indicate that the patient is at risk for developing a disease associated with the above-described kinase.
- the screening and diagnostic methods ofthe invention do not require that the entire kinase subunit DNA coding sequence be used for the probe. Rather, it is only necessary to use a fragment or length of nucleic acid that is sufficient to detect the presence of the kinase subunit gene(s) in a DNA preparation from a normal or affected individual, the absence of such gene, or an altered physical property of such gene (such as a change in electrophoretic migration pattern).
- Prenatal diagnosis can be performed when desired, using any known method to obtain fetal cells, including amniocentesis, chorionic villous sampling (CVS), and fetoscopy.
- Prenatal chromosome analysis can be used to determine if the portion of the chromosome possessing the normal kinase subunit gene is present in a heterozygous state.
- functional kinase or a subunit thereof DNA can be provided to the cells of such patient in a manner and amount that permits the expression ofthe protein provided by such gene, for a time and in a quantity sufficient to treat such patient.
- retrovirus systems can be used, especially modified retrovirus systems and especially he ⁇ es simplex virus systems.
- retrovirus systems can be used, especially modified retrovirus systems and especially he ⁇ es simplex virus systems.
- Such methods are provided for, in, for example, the teachings of Breakefield, X.A. et al, The New Biologist 3:203-218 (1991 ); Huang, Q. et al, Experimental Neurology 775:303-316 (1992), WO93/03743 and WO90/09441. Delivery of a DNA sequence encoding a functional kinase or subunit thereof (as described above) will effectively replace the missing or mutated gene ofthe invention.
- the invention relates to ligands of the above- described kinase.
- the ligand interacts selectively with the kinase.
- Agonists and antagonists ofthe kinase are examples of ligands.
- Antibodies that recognize the kinase or a subunit or functional variant thereof are also ligands.
- the ligand is a selective inhibitor of kinase activity.
- the ligand is a substrate for the above- described kinase.
- Substrates are useful in assay methods for measuring kinase activity.
- Preferred substrates include I ⁇ B ⁇ and peptide or polypeptide fragments thereof.
- the ability of antagonists and agonists ofthe above-described kinase to interfere with or enhance the activity of the above-described kinase can be evaluated in samples containing the above-described kinase.
- An assay for kinase activity in the sample can be used to determine the functionality ofthe protein in the presence of an agent which may act as antagonist or agonist, and thus, ligands that interfere or enhance the activity of the kinase are identified.
- the agents screened in the assays can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents. These agents can be selected and screened 1) at random, 2) by a rational selection or 3) by design using for example, protein or ligand modeling techniques.
- agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to or stimulate/block the activity of the kinase.
- agents may be rationally selected or designed.
- an agent is said to be "rationally selected or designed" when the agent is chosen based on the configuration of the above-described kinase or subunit thereof or known ligand. It is demonstrated herein that Staurosporine and its analogue K252a inhibit the phosphorylation and ubiquitination of I ⁇ B ⁇ in HeLa cell extracts. Additional agents may be designed based on these structures.
- the present invention further provides a method for modulating the activity ofthe above-described kinase in a cell.
- agents including antagonists and agonists as described above
- agent which have been identified to block or stimulate the activity ofthe above-described kinase can be formulated so that the agent can be contacted with a cell expressing the above-described kinase protein in vivo.
- the contacting of such a cell with such an agent results in the in vivo modulation of the activity of the above-described kinase. So long as a formulation barrier or toxicity barrier does not exist, agents identified in the assays described in Section XII below, will be effective for in vivo use.
- the present invention relates to a method of administering the above-described kinase or subunit thereof or a ligand thereof (including kinase antagonists and agonists) to an animal (preferably, a mammal
- NF- ⁇ B is an attractive target for drug design and therapeutic intervention because of its involvement in many pathological conditions such as inflammation, autoimmune disease, cancer and viral infection.
- a number of studies have shown that the inhibition of NF- ⁇ B activity can have profound physiological effects (Kitajima, I., Science 255:1792-1795 (1992); Higgins, K.A. et al, Proc. Natl. Acad. Sci. USA 90:9901-9905 (1993); Kopp & Ghosh, Science 265:956-969 (1994); Reed, M.A. et al, Immunity 2:1-20 (1995); Jung, M. et al, Science 265:1619-1621 (1995); Scheinman, R.I. et al, Science 270:283-286 (1995);
- the present invention relates to a method of using antagonists of the above-described kinase to inhibit activation of NF- ⁇ B.
- NF- ⁇ B may be used to treat disease states characterized by undesired activation of NF- ⁇ B (for example, inflammation, HIV infection, cancer sepsis, psoriasis, restenosis and reperfusion injury).
- the amounts to be administered for any particular treatment protocol can readily be determined.
- the dosage should not be so large as to cause adverse side effects, such as unwanted cross- reactions, anaphylactic reactions, and the like.
- the dosage will vary with the age, condition, sex and extent of disease in the patient, counter indications, if any, and other such variables, to be adjusted by the individual physician.
- Dosage can vary from .001 mg kg to 50 mg/kg ofthe above-described kinase or ligand, in one or more administrations daily, for one or several days.
- the above-described kinase or ligand thereof can be administered parenterally by injection or by gradual perfusion over time. It can be administered orally, intravenously, intraperitoneally, intramuscularly, or subcutaneously.
- Preparations for parenteral administration include sterile or aqueous or non-aqueous solutions, suspensions, and emulsions.
- non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
- Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
- Parenteral vehicles include sodium chloride solution, Ringer's dextrose and sodium chloride, lactated Ringer's, or fixed oils.
- Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives can also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like. See, generally, Remington's Pharmaceutical Science, 16th Ed., Mack
- the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising the above-described kinase or subunit thereof or ligand thereof in an amount sufficient to alter the above-described kinase associated activity, and a pharmaceutically acceptable diluent, carrier, or excipient.
- concentrations and dosage unit sizes can be readily determined by one skilled in the art as described above (See, for example, Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980) and
- the present invention further relates to a method of screening for an agonist or antagonist which stimulates or blocks the activity of the above- described kinase or subunit thereof comprising :
- the sample comprises a cell or cell extract. Any cell or cell extract may be used in the above assay so long as it expresses a functional form ofthe above-described kinase or subunit thereof and the activity can be measured.
- the preferred expression cells are eukaryotic cells or organisms. Such cells can be modified to contain DNA sequences encoding the above-described kinase subunit(s) using routine procedures known in the art. Alternatively, one skilled in the art can introduce mRNA encoding an above-described kinase subunit protein or proteins directly into the cell.
- the sample comprises the above-described purified kinase in an activated state and a substrate.
- MEKKl may be used to activate the kinase.
- the substrate may be I ⁇ B ⁇ or a functional variant thereof or a peptide or a polypeptide that is phosphorylated by the kinase.
- reaction mixtures comprise labeled I ⁇ B ⁇ , an ATP regenerating system, El, an E2, ubiquitin, and a phosphatase inhibitor (Chen et al, Genes and Dev. 9: 1586-1597 (1995)).
- the substrate I ⁇ B ⁇ can contain any detectable label as known to those of ordinary skill in the art, including for example, a radioactive marker, a fluorescent marker, an enzymatic marker, or a chromogenic marker. In the Examples of the current invention, in vitro translated 35 S-labeled I ⁇ B ⁇ was employed.
- the E2 is preferably one ofthe UBC4/UBC5 subfamily.
- the phosphatase inhibitor may be any one of many phosphatase inhibitors known to those of ordinary skill in the art, including but not limited to, okadaic acid, calyculin A, sodium pyrophosphate, sodium molybdate, sodium orthovanadate, or sodium fluoride.
- the phosphatase inhibitor be okadaic acid or calyculin A.
- phosphorylated substrate eg. I ⁇ B ⁇
- phosphorylated substrate is separated from other components of the reaction mixture and quantified by a method appropriate to the label employed.
- samples are analyzed by SDS-PAGE and fluorography. It is to be understood that the use of substrates bearing different labels will necessitate the use of different detection methods such as are known to those skilled in the art.
- kinase inhibition is indicated by a reduction in the amount phosphorylated substrate produced in the test reaction as compared to that produced in a control reaction mixture that does not contain the test substance.
- the non-human animals of the invention comprise any animal having a transgenic interruption or alteration ofthe endogenous ubiquitin dependent kinase subunit gene(s) (knock-out animals) and/or into the genome of which has been introduced one or more transgenes that direct the expression of the above- described kinase.
- non-human animals include vertebrates such as rodents, non-human primates, sheep, dog, cow, amphibians, reptiles, etc.
- Preferred non-human animals are selected from non-human mammalian species of animals, most preferably, animals from the rodent family including rats and mice, most preferably mice.
- the transgenic animals of the invention are animals into which has been introduced by nonnatural means (i.e., by human manipulation), one or more genes that do not occur naturally in the animal, e.g., foreign genes, genetically engineered endogenous genes, etc.
- the nonnaturally introduced genes known as transgenes, may be from the same or a different species as the animal but not naturally found in the animal in the configuration and/or at the chromosomal locus conferred by the transgene.
- Transgenes may comprise foreign DNA sequences, i.e., sequences not normally found in the genome ofthe host animal.
- transgenes may comprise endogenous DNA sequences that are abnormal in that they have been rearranged or mutated in vitro in order to alter the normal in vivo pattern of expression ofthe gene, or to alter or eliminate the biological activity of an endogenous gene product encoded by the gene.
- the transgenic non-human animals of the invention are produced by introducing transgenes into the germline of the non-human animal.
- Embryonic target cells at various developmental stages are used to introduce the transgenes of the invention. Different methods are used depending on the stage of development ofthe embryonic target cell(s).
- Microinjection of zygotes is the preferred method for incorporating transgenes into animal genomes in the course of practicing the invention.
- a zygote a fertilized ovum that has not undergone pronuclei fusion or subsequent cell division, is the preferred target cell for microinjection of transgenic DNA sequences.
- the murine male pronucleus reaches a size of approximately 20 micrometers in diameter, a feature which allows for the reproducible injection of 1 -2 picoliters of a solution containing transgenic DNA sequences.
- transgenic allele demonstrates Mendelian inheritance: half of the offspring resulting from the cross of a transgenic animal with a non-transgenic animal will inherit the transgenic allele, in accordance with Mendel's rules of random assortment.
- Viral integration can also be used to introduce the transgenes of the invention into an animal.
- the developing embryos are cultured in vitro to the developmental stage known as a blastocyst.
- the blastomeres may be infected with appropriate retroviruses (Jaenich, R., Proc. Natl. Sci. (USA) 73:1260-1264 (1976)).
- Infection of the blastomeres is enhanced by enzymatic removal ofthe zona pellucida (Hogan, et al, in Manipulating the Mouse Embryo, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1986)).
- Transgenes are introduced via viral vectors which are typically replication-defective but which remain competent for integration of viral-associated DNA sequences, including transgenic DNA sequences linked to such viral sequences, into the host animal's genome (Janner. et al, Proc. Natl. Acad. Sci. (USA) 52:6927-6931 (1985); Van der Putten, et al, Proc. Natl. Acad. Sci. (USA) 52:6148-6152 (1985)). Transfection is easily and efficiently obtained by culture of blastomeres on a mono-layer of cells producing the transgene-containing viral vector (Van der Putten, et al. , Proc. Natl. Acad. Sci.
- transgenic founder animals produced by viral integration will be mosaics for the transgenic allele; that is, the transgene is incorporated into only a subset of all the cells that form the transgenic founder animal.
- multiple viral integration events may occur in a single founder animal, generating multiple transgenic alleles which will segregate in future generations of offspring.
- transgenes into germline cells by this method are possible but probably occurs at a low frequency (Jahner, D.. et al, Nature 295:623-628 (1982)).
- offspring may be produced in which the transgenic allele is present in all of the animal's cells, i.e., in both somatic and germline cells.
- Embryonic stem (ES) cells can also serve as target cells for introduction ofthe transgenes ofthe invention into animals.
- ES cells are obtained from pre-implantation embryos that are cultured in vitro (Evans, M.J., et al, Nature 292:154-156 (1981); Bradley, M. O., et al, Nature 309:255-258 (1984); Gossler, et al, Proc. Natl. Acad. Sci.
- ES cells which are commercially available (from, e.g., Genome Systems, Inc., St. Louis, MO), can be transformed with one or more transgenes by established methods (Lovell-Badge, R.H., in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, E.J., ed., IRL Press, Oxford (1987), pages 153-182). Transformed ES cells can be combined with an animal blastocyst.
- the ES cells colonize the embryo and contribute to the germline ofthe resulting animal, which is a chimera (composed of cells derived from two or more animals)
- a chimera composed of cells derived from two or more animals
- Bradley. A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, E.J.. ed., IRL Press, Oxford (1987), pages 1 13-151
- offspring may be produced in which the transgenic allele is present in all ofthe animal's cells, i.e., in both somatic and germline cells.
- transgenes ofthe invention may be stably integrated into germ line cells and transmitted to offspring of the transgenic animal as Mendelian loci.
- Other transgenic techniques result in mosaic transgenic animals, in which some cells carry the transgenes and other cells do not. In mosaic transgenic animals in which germ line cells do not carry the transgenes, transmission of the transgenes to offspring does not occur.
- mosaic transgenic animals are capable of demonstrating phenotypes associated with the transgenes.
- Transgenes may be introduced into non-human animals in order to provide animal models for human diseases.
- Transgenes that result in such animal models include, e.g.. transgenes that encode mutant gene products associated with an inborn error of metabolism in a human genetic disease and transgenes that encode a human factor required to confer susceptibility to a human pathogen (i.e., a bacterium, virus, or other pathogenic microorganism)
- a human pathogen i.e., a bacterium, virus, or other pathogenic microorganism
- Transgenically introduced mutations further comprise null ("knock-out") alleles in which a DNA sequence encoding a selectable and/or detectable marker is substituted for a genetic sequence normally endogenous to a non-human animal.
- Resultant transgenic non-human animals that are predisposed to a disease, or in which the transgene causes a disease may be used to identify compositions that induce the disease and to evaluate the pathogenic potential of compositions known or suspected to induce the disease (Berns. A.J.M., U.S. Patent 5,174,986 (Dec. 29, 1992)), or to evaluate compositions which may be used to treat the disease or ameliorate the symptoms thereof (Scott et al, WO 94/12627 (1994)).
- Offspring that have inherited the transgenes of the invention are distinguished from littermates that have not inherited transgenes by analysis of genetic material from the offspring for the presence of biomolecules that comprise unique sequences corresponding to sequences of, or encoded by, the transgenes of the invention.
- biomolecules that comprise unique sequences corresponding to sequences of, or encoded by, the transgenes of the invention.
- biological fluids that contain polypeptides uniquely encoded by the selectable marker of the transgenes ofthe invention may be immunoassayed for the presence of the polypeptides.
- a more simple and reliable means of identifying transgenic offspring comprises obtaining a tissue sample from an extremity of an animal, e.g., a tail, and analyzing the sample for the presence of nucleic acid sequences corresponding to the DNA sequence of a unique portion or portions ofthe transgenes of the invention, such as the selectable marker thereof.
- the presence of such nucleic acid sequences may be determined by, e.g., hybridization ("Southern") analysis with DNA sequences corresponding to unique portions of the transgene, analysis of the products of PCR reactions using DNA sequences in a sample as substrates and oligonucleotides derived from the transgene's DNA sequence, etc.
- Plasmids, proteins and antibodies cDNAs encoding, I ⁇ B ⁇ and its mutants have been described (Brockman, J.A. et al, Mol. Cell. Biol. 75:2809-2818 (1995); Chen, Z.J. et al, Genes & Dev. 9: 1586-1597 (1995)), 35 S-labeled I ⁇ B ⁇ proteins were prepared by in vitro translation in wheat germ extracts (Promega).
- pGEX-2TK-UBCh5 was constructed by PCR using UBCh5 cDNA (provided by Dr. P. Howley) as a template.
- the active site mutants of UBCh5, pGEX-2TK-UBCh5 (C85A) and pGEX-2TK-UBCh5(C85S), were created by site-directed mutagenesis using the Unique Site Elimination (U.S.E) mutagenesis kit (Pharmacia).
- the mutagenic primers (anneal to noncoding strand) were: 5' TTG TGA CCT CAG GAT ATC GAG AGC AAT ACT TCC ATT 3'for C85A, and 5' TTG TGA CCT CAG GAT ATC GAG AGA AAT ACT TCC AT 3' for C85S. All constructs were confirmed by DNA sequencing.
- the appropriate expression constructs were transformed into the E. coli strain
- BL21/DE3 BL21/DE3, and protein expression was induced with 200 ⁇ M IPTG.
- GST fusion proteins were purified using Glutathione-Sepharose (Pharmacia).
- Yeast UBC4 (yUBC4) was expressed in the E. coli AR58 harboring the UBC4 expression vector (pL ⁇ UBC4, provided by Dr. V. Chau). After heat induction (30°C to 42°C), UBC4 was purified by ubiquitin-Sepharose covalent chromatography, followed by gel filtration on FPLC/Superdex-200. Recombinant RelA homodimer was prepared according to Thanos and Maniatis, CeU 50:529-532 (1992)(provided by Dr. J. Hagler).
- (His) 6 MEKKl ⁇ and (His) 6 MEKKl ⁇ (K432M) were purified using Ni- NTA agarose from Sf9 cells infected with baculovirus prepared using the Bac-to- Bac Expression System (GIBCO-BRL Life Technologies).
- pFastBacHT- MEKK1 ⁇ and pFastBacHT-MEKKl ⁇ (K432M) were constructed by subcloning the 1.2 kb Ncol/Xbal coding sequence fragment of pcDNA3-FlagMEKKl ⁇ and pcDNA3-FlagMEKKl ⁇ (K432M), respectively, into the Neo I/Xba I site of pFastBacHTa.
- pGEX-MKK4 was constructed by subcloning the 1.1 kb BamHI (blunt)/Bspl201 (blunt) fragment of pcDNA3-FlagMKK4 containing the MKK4 coding sequence into the Smal site of pGEX-3X (Pharmacia).
- pGEX-JNKl was constructed by subcloning the 1.4 kb Ncol (blunt )/SaII fragment of pSR ⁇ HA- JNK1 containing the JNKI coding sequence into the EcoRI (blunt )/SaII site of pGEX-5X-l (Pharmacia).
- (His) 6 I ⁇ B ⁇ was purified using Ni-NTA agarose from E. coli BL21(DE3)LysS transformed with pRSET-I ⁇ B ⁇ .
- pRSET-I ⁇ B ⁇ was constructed by subcloning the EagI (blunt )/HindIII fragment of pBS-I ⁇ B ⁇ containing the I ⁇ B ⁇ coding sequence into the PvuII/Hindlll site of pRSET A (Invitrogen).
- the E2 enzymes Ubc4 and GST-Ubc5 were prepared as described (Chen, Z.J. et al, Cell 54:853-862 (1996)). Concentrations of recombinant proteins were determined by SDS-PAGE followed by staining with Coomassie blue and comparison with bovine serum albumin standards.
- pCMV5-MEKKl which encodes the C-terminal 672 residues of MEKKl
- pcDNA3-FlagMKK4 and pSR ⁇ HA-JNKl were gifts of Dr. Roger Davis (University of Massachusetts, Worcester) and have been described (Derijard, B. et al, CeU 76: 1025-1037 (1994); Derijard, B. et al, Science 267:682-685 (1995); Whitmarsh, A.J. et al, Science 269:403-407 (1995)).
- pcDNA3-MEKKl was constructed by subcloning the 2.4 kb EcoRI/EcoRI/EcoNI (blunt) fragment of pCMV5-MEKKl encoding MEKKl into the EcoRI/EcoRV site of pcDNA3.
- pcDNA3-Flag MEKK1 ⁇ (K432M) consists of an N-terminal Flag epitope fused to the C-terminal 321 amino acid fragment of MEKKl with the indicated mutation (amino acid numbering according to Lange-Carter, CA. et al, Science 260:315-319 (1993)) and was constructed by polymerase chain reaction (Ausubel, F.M. et al.
- pcDNA3-FlagMEKKl ⁇ was constructed by replacing the 2.1 kb Stul fragment of pcDNA3-FlagMEKKl ⁇ (K432M), which encodes the C-terminal 262 amino acids with the corresponding fragment of pcDNA3-MEKKl .
- K432M Stul fragment of pcDNA3-FlagMEKKl ⁇
- PBS-I ⁇ B ⁇ , pBS- I ⁇ B ⁇ (S32A S36A), pBS-FlagI ⁇ B ⁇ , pBS-FlagI ⁇ B ⁇ (S32A S36A), (PRDII) 2 CAT, (PRDIV) 6 CAT, (CRE) 6 CAT, -HOIFN- ⁇ CAT, and pCMV-lacZ have been described (MacGregor, G.R. and Caskey, C.T., Nucl. Acids Res. 17:2365 (1989);
- HeLa S 3 cell cytoplasmic extracts were prepared by two methods. In the first ("rapid lysis procedure"), mid-logarithmic growth phase HeLa S 3 cells cultured in RPMI 1640 media supplemented with 5% horse serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 ⁇ g/ ml streptomycin were centrifuged at 2,600 x g for 10 min. Cells were resuspended in RPMI media containing 5% horse serum, and then either mock treated or incubated with 1000 U/ml TNF- ⁇ at 37°C At various times, cells were centrifuged at 1 ,000 x g for 1 min at ambient temperature.
- the crude lysate was clarified by centrifugation at 4.600 x g for 10 min at 4°C, and the resulting supernatant immediately frozen at -80°C
- S 100 HeLa S 3 cells were swollen in a hypotonic buffer and lysed, followed by removal of nuclei and centrifugation at 100.000 x g as described (Dignam et al., 1983). The supernatant was then dialyzed extensively against 20 mM Tris (pH 7.5), 0.5 mM DTT. If not employed immediately, the extract was stored at -80 °C
- the active fractions were pooled and applied to FPLC/monoQ which had been equilibrated with Buffer C
- the column was eluted with a linear gradient of 0.15M-0.4M NaCl in Buffer C.
- the fractions containing I ⁇ B ⁇ kinase were concentrated by Centricon-10 and then re-sized on FPLC/Superdex-200 as described above.
- Active fractions were pooled, concentrated and stored at -80°C
- the storage buffer is Buffer A plus 10% glycerol.
- HeLa cell Sl 00 cytoplasmic extract prepared as above, was applied to a Mono-Q anion exchange column.
- the I ⁇ B ⁇ kinase activity was eluted with 0.2-
- KCl 0.3 M KCl in Buffer D (50 mM Tris, pH 7.5, 0.5 mM DTT), and then precipitated with 40% ammonium sulfate.
- the resuspended precipitates were dialyzed against 10 mM K 2 HPO -KH 2 P0 4 (pH 7.0), 0.5 mM DTT, and then applied to a hydroxylapatite column. After elution with 0.2 M K 2 HPO 4 -KH 2 P0 4 (pH 7.0), the kinase-containing fractions were applied to a Superdex-200 gel filtration column equilibrated with 50 mM Tris (pH 7.5), 0.5 mM DTT, and 150 mM NaCl.
- the dialyzed material was then applied to a hydroxyapatite column and I ⁇ B ⁇ kinase was eluted with a linear gradient of 0-0.2 M K 2 HPO 4 -KH,-PO 4 , pH 7.0.
- the active kinase fractions were pooled and applied to a Superdex-200 column in Buffer A containing 150 mM NaCl.
- the high molecular fractions containing the I ⁇ B ⁇ kinase activity was pooled and further fractionated on Mono Q column with a linear gradien of 150-325 mM NaCl in Buffer A. Each fraction was assayed for I ⁇ B ⁇ kinase activity and analyzed for protein content by electrophoresis on 2-15% native gel followed by silver staining.
- phosphorylation of I ⁇ B ⁇ was usually carried out at 37°C for 1 hour in a reaction volume of 10 ⁇ l containing: an ATP regenerating system (50 mM Tris-HCl, pH 7.6, 5 mM MgCI 2 , 2 mM ATP, 10 mM creatine phosphate, 3.5 units/ml creatine kinase, 0.6 units/ml inorganic pyrophosphatase), 0.5 ⁇ l of in vitro translated 35 S-labeled I ⁇ B ⁇ , 2 ⁇ g/ml of recombinant RelA homodimer (to form complex with I ⁇ B ⁇ ), 3 ⁇ M okadaic acid, 60 ⁇ M ubiquitin, 50 nM El (rabbit), I ⁇ M UBC4 (yeast) or GST-UBCh5 (human), and 0.5-3 ⁇ l of I ⁇ B ⁇ kinase containing fractions.
- an ATP regenerating system 50 mM Tris-HCl,
- HeLa fraction I (1 mg/ml) was used as a source of E2s.
- SDS sample buffer was added to quench the reaction, and the samples were analyzed by SDS-PAGE (9%) followed by flourography: Phosphorylation of I ⁇ B ⁇ was quantitated by Phosphorimager analysis. Ubiquitination of I ⁇ B ⁇ has been described previously (Chen, Z.J. et al, Genes & Dev. 9:1586-1597 (1995)).
- the thioester reaction mixtures contain: 50 mM Tris, pH7.6, 0.1 ⁇ M of
- RIPA buffer 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP40, 0.5% deoxycholate, also include 0.1% SDS
- anti-I ⁇ B ⁇ antisera (against the C-terminus of I ⁇ B ⁇ ) were added to the reaction mixture which was then incubated at 4°C for 45 min.
- Protein A-Trisacryl was added to the mixture and the incubation was continued at 4°C for another 45 min.
- the beads were washed three times with RIPA buffer lacking SDS, and three times with Buffer A.
- immunoprecipitates containing I ⁇ B ⁇ (+/- CIP treatment) were washed three times with thrombin buffer (20 mM Tris, pH8.3, 150 M NaCl, 2.5 mM CaCl 2 , and 10% glycerol.), and then treated with 3U of thrombin (Sigma) at 30°C for 2 hours.
- thrombin buffer (20 mM Tris, pH8.3, 150 M NaCl, 2.5 mM CaCl 2 , and 10% glycerol.
- HeLa and L929 cells were maintained in DME media supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin.
- Transfections and virus infections performed in 3.5 cm diameter wells, were conducted as described (Thanos, D. and Maniatis, Cell 77:777-789 (1992)). Cells were typically harvested at 41 to 49 hr posttransfection.
- CAT and ⁇ -galactosidase assays were performed as described (Sambrook, J. et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold S ⁇ ing Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). Protein concentrations were measured by the Bradford method.
- Cell pellets obtained by harvesting 3.5 cm diameter wells were by the addition of 200 ⁇ l of Buffer A (20 mM Tris, pH 7.5, 0.4 M KCl, 4 mM ⁇ -glycerolphosphate, 0.1 mM sodium orthovanadate, 0.1% NP-40, 10% glycerol, 10 ⁇ g/ml leupeptin. 1 mM PMSF, and 1 mM DTT), followed by three freeze/thaw cycles.
- Buffer A (20 mM Tris, pH 7.5, 0.4 M KCl, 4 mM ⁇ -glycerolphosphate, 0.1 mM sodium orthovanadate, 0.1% NP-40, 10% glycerol, 10 ⁇ g/ml leupeptin. 1 mM PMSF, and 1 mM DTT
- the supernatant (320 ⁇ g protein) was incubated with 20 ⁇ l of M2-agarose (IBI- Kodak) in 1 ml of Buffer A with end-over-end rotation for 1 hr at 4°C Resins were then washed three times with Buffer A and once with 0.1 X Buffer A.
- Proteins were electrophoresed by SDS-PAGE and transferred to lmmobilon-NC membranes (Millipore). The membranes were blocked with 5% nonfat milk and probed with rabbit anti-I ⁇ B ⁇ polyclonal antibodies (C21 , Santa
- HeLa cell cytoplasmic extracts or purified I ⁇ B ⁇ kinase (from gel filtration chromatography as described above) was incubated with 0.5 ⁇ l of in v/tro-translated. 35 S-labeled protein in a total volume of 10 ⁇ l containing 50 mM Tris (pH 7.6). 6 mM MgCl 2 , 2 mM ATP, 10 mM phosphocreatine, 3.5 U/ml creatine phosphokinase, and 2.5 ⁇ M okadaic acid.
- Enzyme was incubated with 0.5 ⁇ g (His) 5 I ⁇ B ⁇ in 10 ⁇ l of 50 mM Tris (pH 7.6), 5 mM MgCl 2 , 2.5 ⁇ M okadaic acid, 200 ⁇ M ATP and 5 ⁇ Ci of [ ⁇ - 32 P]ATP. Incubations were carried out at 30°C for 30 min.
- I ⁇ B ⁇ kinase from gel filtration chromatography was treated with MEKK1 ⁇ in 50 mM Tris (pH 7.6), 5 mM MgCl 2 , 2 mM ATP for 30 min at 30°C MEKKI ⁇ -activated I ⁇ B ⁇ kinase was separated from ATP by centrifugal gel filtration on Sephadex G50 and subsequently incubated with or without calf intestinal alkaline phosphatase (CIP) in 50 mM Tris (pH 7.8), 0.1 mM EDTA for 30 min at 30°C l ⁇ B ⁇ kinase was then separated from CIP and MEKK1 ⁇ by chromatography on a Superdex 200 column and assayed for I ⁇ B ⁇ kinase activity in the absence or presence of MEKK1 ⁇ ..
- CIP calf intestinal alkaline phosphatase
- HeLa cell cytoplasmic extracts were separated into fraction I and fraction II by monoQ chromatography. Each fraction alone (lanes 3 & 7) or in combination (lane 8) was assayed for ubiquitination of 35 S-labeIed I ⁇ B ⁇ .
- UBC4 was assayed alone (lane 2) or in combination with fraction II (lane 4).
- a mock E. coli extract (lane 5) or recombinant yeast UBC3 (lane 6) was added together with fraction II. Both UBC4 and UBC3 form thioesters with l25 I -ubiquitin.
- a high molecular weight kinase phosphorylates I ⁇ B at serines 32 and 36
- I ⁇ B ⁇ kinase from monoQ chromatography were pooled and precipitated with 40% ammonium sulfate. The resuspended precipitate was loaded onto a Superdex-200 gel filtration column and fractions were tested for their ability to phosphorylate 5 S-labeled I ⁇ B ⁇ (the generation ofthe slower migrating band, as indicated by p-I ⁇ B ⁇ ).
- the number 670 and 440 above the autoradiogram indicates approximate molecular masses.
- the band above p-I ⁇ B ⁇ is mono-ubiquitinated I ⁇ B ⁇ due to the presence of fraction I in the reaction.
- E2s Only functional UBC4/UBC5 stimulates the phosphorylation of I ⁇ B ⁇ .
- E2s purified from rabbit reticulocytes were tested for their ability to stimulate the phosphorylation of I ⁇ B ⁇ . These E2s include: E2 ]4K (lane 2), E2 17K (lane 3), E2 20 ⁇ (lane 4), E2 25K (lane 5) and E2 35K (lane 6).
- Other E2s that were tested include: purified recombinant human UBC2 (lane 7), yeast UBC4 (lane 8), and human GST-UBC5 ( ⁇ g, lane 9).
- the active site mutants of human UBC5 were tested.
- C85A the active site cysteine at residue 85 (C85) of UBC5 was substituted with alanine.
- C85S C85 of UBC5 was changed to serine. No E2 was added in lane 1.
- the preincubation mixture contains MeUb (40 ⁇ M) instead of ubiquitin.
- the E2-MeUb thioester mixture was then added to the phosphorylation reaction mixture containing 2.4 ⁇ M (lane 7) or 60 ⁇ M (lane 8) of ubiquitin.
- 35 S-labeled I ⁇ B ⁇ was synthesized by in vitro translation, and used as a substrate for phosphorylation (bottom panel, short exposure) and ubiquitination
- lane 5 K252a( 10 ⁇ M) (Calbiochem; Kase, H., et al, Biochem. Biophys. Res. Commun. 742.436 (1987); lane 6: Staurosporine (10 ⁇ M) (Calbiochem; Tomaoki, T., et al, Biochem. Biophys. Res. Commun.
- TPCK and TLCK are alkylating agents that have previously been shown to inhibit the phosphorylation and degradation of I ⁇ B ⁇ in vivo (Beg et al, Mol.
- Staurosporine and K252a inhibit the activation of NF- ⁇ B in vivo by several agonists, including PMA and ionomycin, TNF- ⁇ , and LPS. K252a also inhibits the phosphorylation of I ⁇ B ⁇ by partially purified I ⁇ B ⁇ kinase.
- C HeLa cells were either mock or TNF- ⁇ (5-min) treated, and cytoplasmic extracts prepared by the rapid lysis procedure. Extracts (8 ⁇ g) were then incubated with 35 S-labeled wild-type (WT) or mutant (S32A S36A, M)I ⁇ B ⁇ , or wild-type (WT) or mutant (S63A/S73A, M) c-Jun for 0 or 60 min at 30°C in the presence of 2.5 ⁇ M okadaic acid. Reaction products were subjected to 10% SDS-PAGE and analyzed by autoradiography. Molecular weight markers (in kilodaltons) are indicated to the left. The positions of unphosphorylated (cJun) and phosphorylated (P-cJun) c-Jun are indicated to the right; those for I ⁇ B ⁇ are indicated to the left.
- HeLa (B) and L929(C) cells were transfected with 3 ⁇ g of (PRDII) 2 CAT, 2 ⁇ g pCMV-lacZ, and 4 ⁇ g of pcDNA3-FlagMEKKl ⁇ (K432M) or pcDNA3. Forty to 41 hr posttransfection, some cells were treated with 20 ng/ml mouse TNF- ⁇ (Boehringer) for 8 hr. All cells were harvested 48 to 49 hr posttransfection. CAT activities were normalized to those for ⁇ -galactosidase.
- HeLa cells were transfected with 0.3 ⁇ g of expression vectors for wild ⁇ type (WT) (pCMV4-Flag I ⁇ B ⁇ ) or mutant (M) (pCMV4-Flag I ⁇ B ⁇ [S32A/S36A]) I ⁇ B ⁇ , 3 ⁇ g of pCMV5-MEKKl of pCMV5, and 3 ⁇ g of SP72.
- WT wild ⁇ type
- M mutant
- epitope-tagged I ⁇ B ⁇ was immunoprecipitated, and some samples were treated with calf intestinal alkaline phosphatase (CIP). All samples were then subjected to 10% SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-I ⁇ B ⁇ antibodies.
- CIP calf intestinal alkaline phosphatase
- HeLa cell cytoplasmic extracts were fractionated as described in Experimental Procedures and then chromatographed on (A and B) a Superdex- 200 gel filtration column followed by a (C and D) Mono-Q ion exchange column.
- PKA 0.8 ng, 1 mU, New England Biolabs
- PKC ⁇ 15 ng. Pan Vera
- PKA 0.8 ng, 1 mU, New England Biolabs
- PKC ⁇ 15 ng. Pan Vera
- 35 S- labeled Flag I ⁇ B ⁇ for 30 min at 30°C in the presence of 2.5 ⁇ M okadaic acid.
- An additional incubation (lane 2) contained purified I ⁇ B ⁇ kinase and 35 S-labeled Flag I ⁇ B ⁇ . Reaction products were subjected to 10% SDS-PAGE and analyzed by autoradiography.
- Purified I ⁇ B ⁇ kinase was obtained according to method C above. Fraction 24 from the last MonoQ column was run on 2-15% native gel at 4°C, 45 mV overnight and the protein contents analyzed by silver staining (Figure 19, left page). Shown on the left are the protein markers: 20S: 700 kDa; HSP90: 90 kDa.
- the predominant band beneath the 20S marker on the left panel was excised and run on 12% SDS gel at 25°C, 200V.
- the subunit composition ofthe kinase complex was analyzed by silver staining (Figure 19, right panel) shown on the left are the molecular weights of each individual subunit.
- N-tem inal fragment (residues 5-72) of I ⁇ B ⁇ was expressed in E. coli as a recombinant protein containing a poly-histidine (His6) tag at the N-terminus.
- HeLa cell extract was prepared by hypotonic lysing followed by 100,000 x g centrifugation (S-100). Supematant was collected and a 80% ammonium sulfate precipitation step was followed by dialysis against 20 mM Tris pH 7.6, 1 mM DTT. Cell extract was loaded onto Mono-Q anion exchange column at pH
- kinase was eluted with a 300 mM KCl step gradient. Eluate was further concentrated with 40% ammonium sulfate, then dialyzed against 50 mM HEPES pH 7.6, lmM DTT. This partially purified kinase was activated by incubating with 250 nM El, 750 nM UBC4, 60 ⁇ M ubiquitin, 2.5 mM ATP regeneration system, 3 ⁇ M okadaic acid, for 90 minutes at 3 7°C Peptides from
- Michaelis constant for ATP was determined by first activating the partially purified kinase either with components of the ubiquitination system for
- Each protein subunit shown in Figure 19 was excised from the polyacrylamide gel and digested with trypsin in situ.
- the digested peptides were extracted, separated by HPLC, and microsequenced by tandem mass spectrometry (MS/MS) in the Harvard Microchemistry Facility.
- MS/MS tandem mass spectrometry
- the peptide sequences of p50 and p40 are shown in Figure 21.
- Figure 21 The peptide sequences shown in Figure 21 were used to search the public EST database, and matches are shown in Figure 22 A and B.
- Figure 22 A shows the nucleotide sequence whose translated amino acid sequence contains pep2 (LQEVIETLLSLEK).
- Figure 22B represents the nucleotide sequence whose translated amino acid sequence contains pep4 (TYHALSNLPK).
- nucleic acid sequences shown in Figure 22A and 22B can be radiolabeled and used as probes to obtain the full length clone of p50 and p40, respectively, by screening a cDNA library (ie., HeLa cell cDNA library, for example, see Chen and Pickart, J 73/o/. Chem. 265: 21835-21842 (1990)).
- a cDNA library ie., HeLa cell cDNA library, for example, see Chen and Pickart, J 73/o/. Chem. 265: 21835-21842 (1990)
- TMB 3,3',5,5'-tetramethylbenzidine
- o-PD phenylenediamine dihydrochloride
- HEPES N-[2-Hydroxyethyl]piperazine-N'- [2-ethanesulfonic acid]: DT: dithiothrietol
- ATP Adenosine triphosphate
- SDS- PAGE sodium dodecyl sulfate - polyacrylamide gel electrophoresis
- EDTA ethylenediamine tetra-acetic acid
- TFA trifluoroacetic acid
- PBS phosphate buffered saline
- HPLC High performance liquid chromatography
- FPLC Fast protein liquid chromatography
- SEC size-exclusion chromatography.
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JP09533584A JP2000510328A (en) | 1996-03-19 | 1997-03-19 | Kinase capable of site-specific phosphorylation of IκBα |
AT97916786T ATE265537T1 (en) | 1996-03-19 | 1997-03-19 | KINASE FOR SITE-SPECIFIC PHOSPHORYLATION OF I-KAPPA-B-ALPHA |
KR1019980707386A KR20000064670A (en) | 1996-03-19 | 1997-03-19 | Kinases capable of site-specific phosphorylation of IκBα |
BR9710408A BR9710408A (en) | 1996-03-19 | 1997-03-19 | Kinase capable of ikba specific silicon phosphorylation |
DE69728880T DE69728880T2 (en) | 1996-03-19 | 1997-03-19 | KINASE FOR THE JOB-SPECIFIC PHOSPHORYLATION OF I-kappa B-alpha |
EP97916786A EP0910645B1 (en) | 1996-03-19 | 1997-03-19 | A KINASE CAPABLE OF SITE-SPECIFIC PHOSPHORYLATION OF I-kappa-B-alpha |
AU25317/97A AU728679C (en) | 1996-03-19 | 1997-03-19 | A kinase capable of site-specific phosphorylation of IkBalpha |
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CA (1) | CA2249551A1 (en) |
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CA2263835C (en) | 1996-08-26 | 2012-02-21 | Signal Pharmaceuticals, Inc. | Stimulus-inducible i (kappa)b kinase [ikk] signalsome |
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WO1998037228A1 (en) | 1997-02-25 | 1998-08-27 | The Regents Of The University Of California | IλB KINASE, SUBUNITS THEREOF, AND METHODS OF USING SAME |
US6268194B1 (en) | 1997-02-25 | 2001-07-31 | The Regents Of The University Of California | IKB kinase and methods of using same |
US6242253B1 (en) | 1997-10-09 | 2001-06-05 | Regents Of The University Of California | IkB kinase, subunits thereof, and methods of using same |
-
1997
- 1997-03-19 KR KR1019980707386A patent/KR20000064670A/en not_active Application Discontinuation
- 1997-03-19 DE DE69728880T patent/DE69728880T2/en not_active Expired - Lifetime
- 1997-03-19 WO PCT/US1997/004195 patent/WO1997035014A1/en active IP Right Grant
- 1997-03-19 ES ES97916786T patent/ES2217407T3/en not_active Expired - Lifetime
- 1997-03-19 BR BR9710408A patent/BR9710408A/en not_active IP Right Cessation
- 1997-03-19 US US08/825,559 patent/US6107073A/en not_active Expired - Lifetime
- 1997-03-19 CA CA002249551A patent/CA2249551A1/en not_active Abandoned
- 1997-03-19 AU AU25317/97A patent/AU728679C/en not_active Ceased
- 1997-03-19 AT AT97916786T patent/ATE265537T1/en not_active IP Right Cessation
- 1997-03-19 JP JP09533584A patent/JP2000510328A/en active Pending
- 1997-03-19 EP EP97916786A patent/EP0910645B1/en not_active Expired - Lifetime
-
1999
- 1999-09-24 US US09/406,293 patent/US20020168740A1/en not_active Abandoned
-
2002
- 2002-01-17 US US10/052,005 patent/US6787346B2/en not_active Expired - Fee Related
-
2004
- 2004-03-25 US US10/810,793 patent/US7452977B2/en not_active Expired - Fee Related
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Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
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US6576437B2 (en) | 1996-08-26 | 2003-06-10 | Signal Pharmaceuticals, Inc. | Stimulus-inducible protein kinase complex and methods of use therefor |
US5972674A (en) * | 1996-08-26 | 1999-10-26 | Signal Pharmaceuticals, Inc. | Stimulus-inducible protein kinase complex and methods of use therefor |
US6258579B1 (en) | 1996-08-26 | 2001-07-10 | Signal Pharmaceuticals, Inc. | Stimulus-inducible protein kinase complex and methods of use therefor |
WO1998008955A1 (en) * | 1996-08-26 | 1998-03-05 | Signal Pharmaceuticals, Inc. | Stimulus-inducible i (kappa)b kinase [ikk] signalsome |
US7759476B2 (en) | 1996-08-26 | 2010-07-20 | Signal Pharmaceuticals | Double-stranded RNAS directed to IKK-2 |
US6271199B2 (en) | 1997-02-15 | 2001-08-07 | Millennium Pharmaceuticals, Inc. | Treatment of infarcts |
US6030834A (en) * | 1997-12-30 | 2000-02-29 | Chiron Corporation | Human IKK-beta DNA constructs and cells |
US6077701A (en) * | 1997-12-30 | 2000-06-20 | Chiron Corporation | Ikkβ regulates transcription factors |
WO1999047686A2 (en) * | 1998-03-16 | 1999-09-23 | Cadus Pharmaceutical Corporation | Human mekk proteins, corresponding nucleic acid molecules, and uses therefor |
WO1999047686A3 (en) * | 1998-03-16 | 2000-01-27 | Cadus Pharmaceutical Corp | Human mekk proteins, corresponding nucleic acid molecules, and uses therefor |
US6312934B1 (en) | 1998-03-16 | 2001-11-06 | National Jewish Center For Immunology And Respiratory Medicine | Human MEKK proteins, corresponding nucleic acid molecules, and uses therefor |
US6974677B2 (en) | 1998-03-16 | 2005-12-13 | National Jewish Center For Immunology And Respiratory Medicine | Human MEKK proteins, corresponding nucleic acid molecules, and uses therefor |
US6818427B1 (en) | 2000-10-27 | 2004-11-16 | Millennium Pharmaceuticals, Inc. | MEKK1 molecules and uses thereof |
Also Published As
Publication number | Publication date |
---|---|
EP0910645B1 (en) | 2004-04-28 |
DE69728880D1 (en) | 2004-06-03 |
JP2000510328A (en) | 2000-08-15 |
US20040161814A1 (en) | 2004-08-19 |
ATE265537T1 (en) | 2004-05-15 |
US7452977B2 (en) | 2008-11-18 |
BR9710408A (en) | 1999-08-17 |
US20020151022A1 (en) | 2002-10-17 |
ES2217407T3 (en) | 2004-11-01 |
US6787346B2 (en) | 2004-09-07 |
US20020168740A1 (en) | 2002-11-14 |
KR20000064670A (en) | 2000-11-06 |
EP0910645A1 (en) | 1999-04-28 |
AU2531797A (en) | 1997-10-10 |
AU728679B2 (en) | 2001-01-18 |
DE69728880T2 (en) | 2005-04-28 |
AU728679C (en) | 2002-03-21 |
US6107073A (en) | 2000-08-22 |
CA2249551A1 (en) | 1997-09-25 |
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